BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 967-975
Vol. 173, No. 3, 1990 December 31, 1990
NUCLEOTIDE SEQUENCE OF THE COMPLEMENTARY DNA FOR TURKEY GROWTH HORMONE
DOUGLAS N. FOSTER , , SUNG U. KIM 2, JOHN J. ENYEART 2, and LINDA K. FOSTER 1
IDepartment of Poultry Science, Laboratories of Molecular and Developmental Biology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691 2Department Of Pharmacology, College of Medicine, The Ohio State University, Columbus, OH 43210
Received November 9, 1990
SUMMARY
Near-full length complementary DNA (eDNA) clones encoding turkey
growth hormone (GH) have been isolated from a pituitary library.
The longer
of the two turkey GH cDNA clones that were sequenced is 803 base pairs (bp) in length and contains 41 nucleotides of the 5'-untranslated region (UTR), an open reading frame of 648 bp that encodes a 25 amino acid leader polypeptide segment as well as a 191 amino acid mature turkey GH protein, and a 3'-UTR that is 92 bp long followed by a 22 bp poly A tract.
Comparison of the turkey
GH nucleotide sequence to that of other avian GH clones shows the coding region to be greater than 93~ homologous while the homology to mammalian GH sequences is between 68 and 78~.
Northern blot analysis showed an approximate
800 bp turkey GH processed mRNA transcript that hybridized to the turkey GH cDNA probe.
A large up-regulation of turkey GH transcription occurred when
intact cultured pituitaries were treated with 1 nM human growth hormone releasing hormone but only modest changes were observed when cultures were treated with thyroid releasing hormone or somatostatin.
©1990Academicp~esstInc.
Although the primary amino acid sequence of the chicken growth hormone (GH) has been reported (i), only recently has the nucleotide sequence been published (2).
To date, duck GH is the only other avian GH that has been
cloned and nucleotide sequenced (3).
We report here the isolation, cloning
and structural analysis of complementary DNA (cDNA) encoding turkey GH.
The
cloning of a GH cDNA from a third avian species adds to the existing database
*To whom correspondence should be addressed.
967
FAX 216-263-3675.
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
present for this biologically important molecule and permits the comparison of its amino acid and nucleotide sequences with those from a number of other mammalian and non-mammalian vertebrate species. With the potential of expressing large amounts of recombinantly derived turkey GH using eukaryotic expression vector systems (4,5) it should be possible to learn more about how GH regulates growth, metabolism and mineral balance in the turkey.
Furthermore, with the use of the cloned turkey GH cDNA
and genomic sequences (when they become available) it should be possible to learn how the synthesis of GH is regulated by hormones which enhance or inhibit GH gene transcription.
MATERIALS AND METHODS Isolation and culturing of pituitaries The anterior pituitaries were removed from 40 eight-week-old turkeys at the OARDC turkey research facilities. Organs were placed into sterile medium 199 plus antibiotics on ice. Extraneous tissue was removed and the pituitaries were minced and placed in static culture in fresh 199 medium plus antibiotics and i0 nM chicken gonadotrophin releasing hormone i (GnRH) for i hr at 41 ° C. (It has been reported that GnRH causes increased levels of GH secretion in goldfish (6)). Following, treatment glands were snap-frozen in liquid nitrogen. RNA isolation and eDNA library construction Total cellular RNA was isolated using the guanidinium isothiocyanate method: (7). Poly A containing mRNA was isolated by oligo dT cellulose chromatography as previously described (8). Turkey pituitary RNA that was double passed over oligo dT cellulose was used to prepare double-strand eDNA using the protocols described in the manufacturer's kit (Amersham, Arlington Heights, IL). The resulting methylated eDNA was blunted ended with the large Klenow fragment of DNA polymerase i, and EcoRl linkers were added. Following EcoRl digestion, the linkered eDNA was size fractionated by Sepharose 4B chromatography then ligated to EcoRl phosphatased lambda Zap II vector DNA (Stratagene Inc., La Jolla, CA)with recombinant constructs propagated in XL-I Blue E.coli (Stratagene Inc., La Jolla. CA). The primary library was titered and stored over chloroform. The IXI05 member library was 88% recombinant as determined by the chromogenic indicator X gal (9). eDNA cloning and sequencing strategy Approximately 50,000 plaques of the amplified turkey pituitary eDNA library were screened as previously described (i0). Duplicate nitrocellulose lifts were prepared as described (ii) then prehybridized (16 hrs) and hybridized (16-24 hrs) in non-fat dry milk/formamide solution (12). Filters were probed with a gel purified chicken growth hormone 807 bp eDNA insert (2) that was 32p labeled using the random primed oligonucleotide procedure (13). Several positive clones were selected for subsequent rounds of screening and four positive clones were plaque purified. Bluescript plasmids containing turkey GH cDNA inserts were excised from the lambda Zap II phagemid vector with helper virus (Stratagene Inc., La Joll~, CA). DNA from the four plasmids was Eco____Rldigested, separated by agazose gel electrophoresis, ethidium bromide stained, and Southern transferred to a nylon membrane (9). The filter was hybridized to 32p labeled chicken GH insert as above. All four plasmids contained inserts that hybridized to the probe. Clones i and 4 contained the two largest inserts (approximately 800 bp) and were selected for subsequent sequence analysis using the dideoxy chain termination method (14), primers KS and SK specific for the Bluescript vector (Stratagene Inc., La Jolla, CA) and the modified T7 polymerase, Sequenase (U.S. Biochemical, Cleveland, OH). Sequence data were compiled and analyzed using the MicroGenie Program (Version 6.0; Beckman Instruments, Palo Alto, CA).
968
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Northern blot analysis Forty pituitaries were removed from nine-week-old turkeys and placed in DMEM medium on ice. Pituitaries were separated into four equal groups and placed into culture dishes containing i0 ml DMEM plus antibiotics and incubated for 1 hr at 41 ° C with the following: I) medium alone, (control); 2) 1 n M h u m a n growth hormone releasing hormone (GRF, Peninsula Laboratories, Belmont, CA); 3) I0 nM somatostatin (SRIF, Sigma Chemical Co., St. Louis, MO); 4) I0 nM thyroid releasing hormone (TRH, Sigma Chemical Co., St. Louis, Mo). After incubation, the glands were snap-frozen in liquid nitrogen. Turkey brain and liver tissue were used as negative control tissue. Total cellular RNA was extracted from the tissues (approximately 150 mg) using the protocols previously described (7) then phenol extracted and ethanol precipitated. Ten micrograms of total cellular RNA from each of the samples was denatured in gel-running buffer (9) at 55 ° C for 15 minutes, fractionated by formaldehyde agarose gel electrophoresis and transferred onto a Zeta-probe nylon membrane (Bio-Rad, Richmond, CA) as described (9). The filter was baked as above, then prehybridized for 6-8 hrs at 42 o C in a diethylpyrocarbonate treated solution of non-fat dry milk that contained 509 formamide plus 19 SDS (12) and probed with the 32p labeled chicken GH eDNA insert (6-7XI0 6 dpm/ml). After overnight hybridization at 42 o C, the filter was washed in 2X SSC (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0) plus 0.19 SDS at 55 ° C, then 0.2X SSC plus 0.19 SDS at 60 ° C, and finally at 0.02X SSC plus 0.19 SDS at 65 ° C (45 minutes for each wash). The filter was exposed to Kodak XAR film for 3 hrs at -80 ° C. The level of probe hybridization to specific mRNA bands was quantified from autoradiographs using a scanning densitometer (E-C Apparatus, St. Petersberg, FL).
RESULTS Identification of cDNA coding for turkey growth hormone
A turkey pituitary
cDNA library was screened for GH using a purified heterologous 32p labeled chicken GH cDNA insert as probe.
Four plaque purified clones remained
positive after several rounds of screening.
Southern blot analysis of the
four recombinant plasmid cDNAs showed that all four eDNA inserts hybridized intensely to the probe.
The largest of the clones (i and 4) were selected for
nucleotide sequence analysis.
Figure 1 shows the composite nucleotide
sequence of the two different turkey GH eDNA clones (i and 4).
The turkey GH
nucleotide sequence contains a unique BamHl site that bisects the clones so that deletion of the EcoRl - BamHl fragment could produce sequence information from the internal portions of each clone.
Clone 1 lacked 7 bp (CAAAGGA) at
the 5 t end of the sequence shown in Figure 1 while it was determined that clone 4 missed the last two bp at the 3' end of the sequence (TG) as well as the poly A containing region.
Otherwise these two different clones were found
to be identical when overlapping sequences were compared.
In addition to the
sequence for the GH coding region, turkey GH clone 1 contains 41 bp of the 5'untranslated region (UTR), 92 bp of 3t-UTR and a 22 bp poly A tract while clone 4 contains 48 bp of 5' UTR, 90 bp of 3'-UTR and no poly A tract. poly A signal sequence AATAAA resides 14 bp from the poly A tract.
The
The turkey
GH eDNA clones 1 and 4 encode a 25 amino acid leader polypeptide and a 191 amino acid mature protein.
The deduced relative molecular weight (Mr) of the
mature protein is 22,272 daltons.
969
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-24 C•AAGGA•AGCTCACATTT•AACATC•TCCTGCATTTTCTCATCTTTCTGATTTATT•T•ATTGAACAAGGGAGAAAGATC ATG GAT M
D
88
-20 -i0 -i i TGC TAC AGG AAG TAT GCA GCT GTC ACT TTG ACC ATT TTG TCT GTG TTT CTG CAT CTT CTT CAT ACT TTC C Y R K Y A A V T L T I L S V F L H L L H T F
157
I0 20 CCA GAT GGA GAA TTT CTC ATG CAG GGT TGT CCA GAG TGC AAG CTA GGG GAG AAC AGG TTC TTT TCA AAA P
D
G
E
F
L
M
Q
G
C
P
E
C
K
L
G
E
N
R
F
F
S
K
30 40 CCA GGA GCC CCC ATT TAC CAG TGC ACT GGG TGC TGT TTC TCC CGG GCC TAT CCT ACT CCA ATG AGA TCC
226
P
G
A
P
I
Y
Q
C
T
G
C
C
F
S
R
A
Y
P
T
P
M
R
S
50 60 70 AAG AAG ACC ATG CTT GTT CCA AAG AAC ATT ACA TCG GAA GCA ACG TGC TGT GTA GCA AAG GCT TTC ACC
295
K
K
T
M
L
V
P
K
N
I
T
S
E
A
T
C
C
V
A
K
F
T
90
80
364
A
AAG ATT ACC CTT AAG GAC AAT GTG AAG ATA GAG AAC CAC ACA GAG TGT CAC TGC AGT ACC TGC TAC TAT K
I
T
L
K
D
N
V
K
I
E
N
H
T
D
C
H
C
S
T
C
Y
Y
96 CAT AAA TCT TAA AGCCTGTCCCTTTGCTAATGATCAAGAACAACGGTGAATGAAATATTTGTTGTTCAGCTTTTACAGCACCGCTGT
433
H
K
S
END
520
GTATAAT•TTGTGTTTT•TGGTCAAGACACCGAGTAGACTTTTGAATGAGATGGATGG•TGTTTTATTTCCTCTTTG•TT•TTCATGCATT
611
TAAGTAAGTTTAACTATTTCCATTAGGGATTAGATGTAGCCCTTGCATGACAACCATAAG•TTGATCTGTTTTTAAAATAAACTGCTAGAT
702
AAATTGTC [POLY A (45)]
Figure I.
The
Nucleotide sequence and corresponding amino acid coding sequence of turkey GH. The amino acids for the signal polypeptide are indicated by negative numbers while those for the mature GH protein are indicated by positive numbers.
size,
transcript Results
was
regulation
indicate
hybridized
specificity
that a s i n g l e p r o c e s s e d
to the
of p i t u i t a r i e s
and tissue
determined by northern blot
t u r k e y GH p r o b e
transcription
(lane 2) w h i l e
down regulate
t u r k e y GH m R N A r e s p e c t i v e l y
brain
(lane
5) a n d l i v e r
c D N A as e x p e c t e d
transcript
for c o n t r o l
with GRF caused a greater
of the
analysis
t h a n 20 f o l d
T R H a n d SRIF a p p e a r e d (lanes
that the p r o b e was
in F i g u r e
of a p p r o x i m a t e l y
pituitaries
induction
3 and 4).
Treatment
of t u r k e y GH
slightly
up a n d
RNA from turkey
to the l a b e l e d
tissue
2.
800 bp
(lane I).
to o n l y
(lane 6) d i d n o t h y b r i d i z e
indicating
t u r k e y GH m R N A
as s h o w n
turkey
GH
specific.
DISCUSSION Two d i f f e r e n t been
isolated
near
sequence
has b e e n
compared
to s e q u e n c e
high
degree
when
determined. data
When
growth hormone
The
compared
the c o d i n g
observed
t u r k e y GH also to the m a m m a l i a n
complementary
gland cDNA library region
for GH f r o m o t h e r a v i a n
of s e q u e n c e h o m o l o g y
respectively). 78%)
full-length
from a turkey pituitary
shares
of the t u r k e y GH is
species
(2,3)
there
(96% a n d 93% for c h i c k e n considerable
species human
970
DNAs have
and their n u c l e o t i d e
sequence
(15), b o v i n e
is a
and duck
homology (16),
rat
(68%(17),
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
3
4
800 bp -
Figure 2.
and mouse
Regulation of turkey GH transcription was performed by northern blot analysis. Total cellular RNA was prepared from nine week-old turkey pituitaries that received either no in vitro treatment (lane I); or I nM human GRF (lane 2); i0 nM SRIF (lane 3); i0 nM TRH (lane 4). Total RNA was also prepared from turkey liver (lane 5) and brain (lane 6) to serve as negative controls. Samples were fractionated by formamide gel electrophoresis, transferred to a nylon membrane, and hybridized to a 32p labeled turkey GH cDNA. The membrane was washed successively in 2X, 0.2X and 0.02X SSC at 55-65C, prepared for autoradiography and exposed overnight to Kodak XAR-5 film.
(18), but drops to 58~ homology when compared to salmon GH (19).
The nucleotide
sequence
chicken sequence
for the leader polypeptide
(97~ homology)
even less for the mammalian
species examined
82~) but only 46-54~ for the mammalian
(50~-58~).
species compared and 33~ for salmon GH.
of the chicken and duck (61~-78~)
and mammals examined
of the turkey GH cDNA agrees with other avian species but differs
contain additional There appears observed
The 5'-untranslated
homology with Chicken and duck (78-
the 3~-UTR of the turkey GH shares even less homology
(15,16,17,18)
(38~-42~).
from the salmon species
3F-UTR sequences
perhaps
~20 K variant'
important
The length
(19,20) which
for mRNA stability.
in the turkey GH cDNA as
of human GH.
thought to be the result of an alternative
than the 5'-UTR
(2,3) and mammalian
to be no smaller splicing variant
in the so-called
to the
but is less conserved for the duck (81~) and
region of turkey GH shares considerable
Overall,
is almost identical
The human variant
splicing of the pre-mRNA
is
transcript
(21). Figure 3 compares
the leader polypeptide
GH with several other species. 96~ homology mammalian
to chicken
species
is very homologous
The 25 amino acid signal polypeptide
shares
(2) and 83~ to duck (3), but only 24-32~ with the
that were compared to chicken
mammalian GH proteins
and mature protein for turkey
(15,16,17,18).
(96~) and duck (95~),
(15,16,17,18,22,23,24)
971
The turkey GH apoprotein 75-80~ homologous
while 69~ homologous
to
to the blue
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-24
-20
-i0
Me••As••Cy•-Tyr•Arg-Lys•Tyr•A•a-A•a••a••Thr-Leu•Thr-I•e-Leu•Ser-Va•-Phe-•eu-His-Leu-Leu-His•Thr
chicken bovine human mouse
-
rat
Tyr Tyr Tyr
.
.
. . .
. . .
. . .
. . X X
. . .
. . .
. . .
. . .
. -
. . . Val-Thr Val-Met Val-Mec X X
.
. . -
-
Leu . . . Met Met-Val X X
.
.
Ile Val lie
-
-
Set Ser Set Set X
X
Ar K X
. X
X
X
X
Ile X
X
X
X
X
X
lie X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
equine
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
1
10
20
Iq~e~T~m~y~1u~Phe~Leu~t~e~n-G1y-C~s~Pr~-G1u~Cys~Lym-Leu-~y~u~Asn~Arg-I~he~Fbe~Ser-Lys .
.
.
human
Ala
-
mouse
Leu
-
rat porcine
Leu X
X
ovine
.
equine
.
.
-
Val
X
X
-
Asp
-
X
Asp-Leu-Ile-ne X X Thr
X
.
.
ASh Gln . . .
.
Thr
.
X
X
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. . Asp
-
.
.
Asp
.
.
.
. .
Thr . Thr-Thr
.
-
ne-ne
-
.
.
.
.
.
.
Thr
. .
chicken bovine
l'ro-Cly-AlaAsp .
human mouse
. . . . Leu . .
. .
.
.
rat porcine
Leu Leu
. .
. .
. .
ovlne
-
equine
Leu
. .
.
.
.
Gin .
.
.
Asp
.
-
.
.
VaI
.
Leu . . . . .
.
-
-
. . .
.
. .
.
Met Met
. .
Met
.
-Cys. .
Phe-Ser . .
. .
. .
. .
. .
chicken
. . .
rat porcine ovine equine
.
-Leu-Val-
.
.
-
Pro
Lys
-
Lys-Tyr
-
Lys Lys
-
Lys-Tyr Lys-Tyr
-
Lys Art
-
Lys -Tyr Lys-Tyr
-
-Ar&-Ala-Tyr-Plco-1[hr. . . . .
. .
. .
. .
. .
. .
chicken
.
. .
.
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
A1a Ala
.
.
.
.
.
.
.
.
.
.
.
Ala
Lys
.
.
.
.
.
.
.
.
.
.
.
Ala
.
.
.
.
.
.
.
Gln . .
.
.
Val . . .
.
.
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. -
Val-Met-Giy-Gly-Phe -
porcine ovine
Val -Met -Gly Val-Met-Gly
-
equine
Val-Met-Gly
Figure 3.
-
- Pal Ala-Arg-Vai Ala-Arg-Val
Ala-Arg-Val Arg-Vai lie
-
Leu
Art
-Cya - Cym=Val-kla-Lys
.
.
Set . .
. .
.
. .
.
-Ala-
Phe -l~,r .Lyz- Ile
.
.
.
Ala
.
. .
. .
.
Set -Tyr -Asn-Arg.Val Ala
.
.
.
. .
. .
. .
. .
. .
. .
. .
.
.
.
.
Ala Aia
.
.
.
.
.
.
.
.
.
.
.
Ala
.
Set
.
Set
.
.
.
.
.
.
-
-
.
-
lle-Arg-Val
90
. . .
96
-Thr -Asp -Cys -His -Cys- Set -Thr- Cys .Tyr. Tyr .His .Lys. Set
. . . . . . . . .
.
Glu
.
.
Ala Glu . .
. . .
. .
.
.
. . .
. . .
. .
. .
. .
. .
Olu Glu
. .
. .
.
.
.
.
Gin
-
Tyr
. .
.
.
. . .
. . .
. . .
. . .
. . . .
. . .
. . . .
. . .
. . . .
. .
. . .
. . .
. . .
.
. .
. . His
-
Ile
The primary amino acid structure of turkey GH compared to other avian, mammalian and non-mammalian GH sequences. The leader sequences include amino acids -95 to -I and the mature protein include amino acids 1 to 191. Dashes indicate the same amino acids as for turkey GH. Lack of a comparable amino acid is shown by X. Comparisons of known amino acid sequences are for chicken (2), duck (3), bovine (16), rat (17), mouse (18), equine (22), human (15), and salmon (19).
(25) but only 55% homologous (26,27,28).
Surprisingly,
(89%) to the sea turtle
(29).
to salmon (19) and 40% for other fish turkey GH shows a high degree of homology
The four cysteine
181, and 189) which are important to structural same in all species
that were compared.
that are invariant regardless
(positions
115-120;
82-87;
evolutionarily
(positions
of the species compared
153-162) which suggest that these regions have been
site at the carboxyl
Asn-X-Asn)
binding or
functions.
When comparing avian GH proteins
there appears
to be no putative
termini of the turkey GH protein
as is found for chicken and duck (Asn-X-Thr).
of interest to utilize
53, 164,
folding were found to be the
conserved and might be important for ligand/receptor
specific biological
glyeosylation
residues
Figure 3 shows that there are regions
of block homology
may possess
Lys
.
Thr - Leu- Lys -Asp -ASh- Val -Lys -lle-Glu-Asn-Hls Val-Met-Gly Arg-Val . . . . Val-Met-Gly Val -Met-Gly
-
70
Pro - Lys -ASu- Ue -Thr - Set -Glu=Ala-Thr
.
.
.
Phe
Leu Ala
Met
. .
human mouse rat
Gln
pro -~let.~g.~r. Ala
. .
80 bovine
-
-
60
Lys -Thr -t~t
human mouse
Glm
Mec
50 ~vine
Lys-Tyr
~.0
Pro- I le -Tyr -ciu-Cys-Thz-Giy-Cys . . . . . Met: .
. .
-
-
.
.
.
Ly$
.
30
188-190;
-
. X
bovine
species
Gin
-
ovine
chicken
shark
-
porcine
X
.
lie Ile-Phe Ile
site-directed mutagenesis
techniques
(positions It would be
in order to change
turkey GH amino acid position 190 to either a Thr or Set to allow for posttranslational
glycosylation.
The production o f b o t h
972
recombinantly
derived
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
glycosylated as well as non-glyeosylated turkey GH in eukaryotic expression systems (4,5) would allow one to test the biological activity of the two GH isoforms. A number of peptide and steroid hormones acting alone and synergistically are known to regulate GH transcription and secretion.
The
northern blot data shown in Figure 2 (lane 2) reveals a large (greater than 20 fold by densitometry) and rapid (one hour) up-regulation of the steady-state levels of turkey GH mRNA suggesting that expression is at least in part regulated at the transcriptional level.
The observed increase in accumulated
levels of GH mRNA may be the result of increased transcription, message stability, or both.
A similar (albeit much lower) increase in mRNA levels
upon GRF induction has been observed in cultured bovine pituitary cells (30). SRIF administration has been shown to lower GH secretion in avian species (34,35,39) and the data in Figure 2 (lane 3) shows that SRIF marginally downregulates turkey GH transcription (.77 fold).
TRH has been reported to cause
increased GH secretion (31,32,33,34,35,36,37) in avian species and was shown in Figure 2 (lane 4) to cause a slight increase (1.85 fold) in turkey GH transcription as previously reported (38).
In addition to hypothalamic
peptides a number of peripheral hormones participate in the regulation of GH synthesis and secretion.
Insulin-like growth factor-i which mediates many of
the actions of GH, feeds back on the pituitary and hypothalamus to inhibit GH gene expression and secretion (36, 40).
Thyroid hormones, glucocorticoids and
retinoic acid act alone or in concert to enhance GH gene transcription (41,42,43,44).
The cloning of the turkey GH eDNA along with genomic sequences
in the near future should allow a better understanding of the complex hormonal control of GH gene expression.
ACKNOWLEDGMENTS The authors thank Dr. Mark Heiman, Eli Lilly and Company, Lilly Research Laboratories, Greenfield, IN, for the gift of human growth hormone releasing factor.
These sequence data will appear in the EMBL/Genbank/DDBJ Nucleotide
Sequence Data bases under the assession number M 33697.
Salaries and research
support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No. 300-90.
REFERENCES i.
2.
Souza, L.M., Boone, T.C., Murdock, D., Langley, K., Wypych, J., Fenton, D., Johnson, S., Lai, P.H., Everett, R., Hsu R.- Y., and Bosselman R. (1984) J. Exp. Zool. 232,465-473. Lamb, I.C., Galehouse, D.M., and Foster, D.N. (1988) Nuc. Acids Res. 16,9339.
973
Vol. 173, No. 3, 1990 3.
4. 5. 6. 7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
20.
21. 22.
23 24 25 26 27 28. 29. 30. 31. 32. 33. 34. 35. 36.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chen, H.-T., Pan, F-M., and Chang, W-C. (1988) Biochim. Biophys. Acta 949,247-251. Leung, F.C., Jones, B., Steelman, S.L., Rosenblum, C.I., and Kopchick, J.J. (1986) Endocrinol. 119,1489-1496. Luckow, V.A., and Summers, M.D. (1988) Bio/Technol. 6,47-55. Marchant, T.A., Chang, J.P., Nahorniak, C.S., and Peter, R.E. (1989) Endocrinol. 124,2509-2518. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159. Aviv, H., and Leder, P., (1972) Proc. Natl. Acad. Sci., USA, 69,14081412. Maniatis, T., Fritsch, E.F., and Sambrook, J., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y Benton, W.D., and Davis, R. (1977) Science 196,180-182. Foster, D.N. (1987) Ohio Acad. Sci. 87,162-165. Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H. (1984) Gene. Anal. Techniques 1,3-8. Feinberg, A.P., and Vogelstein, B. (1983) Anal. Biochem. 132,6-13. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci., USA, 74,5463-5467. Martial, J.A.O., Hallewell, R.A., Baxter, J.D., and Goodman, H.M. (1979) Science 205,602-607. Miller, W.L., Martial, J.A., and Baxter, J.D., (1980) J. Biol. Chem. 255,7521-7525. Seeburg, P.H., Shine, J., Martial, J.A., Baxter, J.P., and Goodman, H.M. (1977) Nature 270,486-494. Linzer, D.I.H., and Talamantes, F. (1986) J. Biol. Chem. 260,9574-9579. Sekine, S., Mizukami, T., Nishi, T., Kuwona, Y., Saito, A., Sato, M., Itoh, S., and Kawauchi, H. (1985) Proc. Natl. Acad. Sci. USA 82,43064310. Nicoll, C.S., Steiny, S.S., King, D.S., Nishioka, R.S., Mayer, G.L., Eberhardt, N.L., Baxter, J.D., Yamanaka, M.K., Miller, J.A., Seilhamer, J.J., Schilling, J.W., and Johnson, L.K. (1987) Gen. Comp. Endocrinol. 68,387-399. DeNoto, F.M., Moore, D.D., and Goodman, H.M. (1981) Nuc. Acids Res. 9,3719-3730. Zakin, M.M., Pokus, E., Langton, A.A., Ferrara, P., Santome, J.A., Dellacha, J.M., and Paladini, A.C. (1976) Int. J. Pept. Protein Res. 8,435-444. Li, C.H., Chung, D., Lahm. H.-W., and Stein, S. (1986) Arch. Biochem. Biophys. 245,287-291. Li, C.H., Gordon, D., and Knorr, J. (1973) Arch. Biochem. Biophys. 156,493-508. Yamaguichi, K., Yasuda, A., Lewis, U.J., Yokoo, Y., and Kawauchi, H. (1989) Gen. Comp. Endocrinol. 73,252-259. Sato, N., Watanabe, K., Murata, K., Sakaguchi, M., Kariya, U., Kimura, S., Nonaka, M., and Kimura, A. (1988) Biochem. Biophys. Acta 949,35-42. Watahiki, M., Tanaka, M., Masuda, N,, Yamakawa, M., Yoneda, Y., and Nakashima, K. (1988) Gen. Comp. Endocrinol. 70, 401-40. Kawauchi, H., Moriyama, S., Yasuda, A., Yamaguchi, K., Shirahata, K., Kubota, J., and Hirano, T.K (1986) Arch. Biochem. Biophys. 244,542-552. Yasuda, A., Yamaguchi, K., Papkoff, H., Yokoo, Y., and Kawauchi H. (1989) Gen. Comp. Endocrinol. 73,242-251. Tanner, J.W., Davis, S.K., McArthur, N.H., French, J.T., and Welsh, T.H., Jr. (1990) J. Endocrinol. 125,109-115. Harvey, S. (1990) J. Endocrinol. 126,75-81. Harvey, S., Lea, R.W., and Ahene, C. (1990) J. Endoerinol. 126,83-88. Harvey, S. (1990) J. Endocrinol. 125,345-358. Scanes, C.G., and Lauterio T.J. (1984) J. Exp Zool. 232,443-452. Scanes, C.G. (1987) In Critical Reviews in Poultry Biology (R.R. Dietert, Ed.), Vol. i, pp. 51-105, CRC Press, Boca Raton, FL. Perez, F.M., Malamed, S., and Scanes, C.G. (1989) Gen. Comp. Endocrinol. 73,12-20.
974
Vol. 173, No. 3, 1990
37. 38. 39. 40. 41. 42. 43. 44.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Harvey, S., Foltzer-Jourdainne, C., Karmann, H., and Mialhe, P, (1988) Gen. Comp. Endocrinol. 70,374-381. Seo H. (1985) In The Pituitary Gland (H. Imura, Ed.) pp. 57-82, Raven Press, New York, NY. Harvey, S., and Scanes, C.G. (1987) Comp. Biochem. Physiol. 87A,315-318. Buonomo, F.C., Lauterio, T.J., Baile, C.A., and Daughaday, W.H. (1987) Gen. Comp. Endocrinol. 66,274-279. Seeburg, P. (1980) In Polypeptide Hormones (R.F. Beers, Jr., and E.G. Bassett, Ed.) pp. 19-31, Raven Press, New York, NY. Tushinski, R.J., Sussman, P.M., Yu, L.-Y., and Bancroft, F.C. (1977) Proc. Natl. Aead. Sci. USA 74,2357-2361. Nyborg, J.K., Nguyen, A.P., and Spindler, S.T. (1984) J. Biol. Chem. 259,12377-12381. Bedo, G., Santisteban P., and Aranda, A. (1989) Nature 339,231-234.
975
Vol. 173, No. 3, 1990 December 31, 1990
NUCLEOTIDE SEQUENCE OF THE COMPLEMENTARY DNA FOR TURKEY GROWTH HORMONE
DOUGLAS N. FOSTER , , SUNG U. KIM 2, JOHN J. ENYEART 2, and LINDA K. FOSTER 1
IDepartment of Poultry Science, Laboratories of Molecular and Developmental Biology, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691 2Department Of Pharmacology, College of Medicine, The Ohio State University, Columbus, OH 43210
Received November 9, 1990
SUMMARY
Near-full length complementary DNA (eDNA) clones encoding turkey
growth hormone (GH) have been isolated from a pituitary library.
The longer
of the two turkey GH cDNA clones that were sequenced is 803 base pairs (bp) in length and contains 41 nucleotides of the 5'-untranslated region (UTR), an open reading frame of 648 bp that encodes a 25 amino acid leader polypeptide segment as well as a 191 amino acid mature turkey GH protein, and a 3'-UTR that is 92 bp long followed by a 22 bp poly A tract.
Comparison of the turkey
GH nucleotide sequence to that of other avian GH clones shows the coding region to be greater than 93~ homologous while the homology to mammalian GH sequences is between 68 and 78~.
Northern blot analysis showed an approximate
800 bp turkey GH processed mRNA transcript that hybridized to the turkey GH cDNA probe.
A large up-regulation of turkey GH transcription occurred when
intact cultured pituitaries were treated with 1 nM human growth hormone releasing hormone but only modest changes were observed when cultures were treated with thyroid releasing hormone or somatostatin.
©1990Academicp~esstInc.
Although the primary amino acid sequence of the chicken growth hormone (GH) has been reported (i), only recently has the nucleotide sequence been published (2).
To date, duck GH is the only other avian GH that has been
cloned and nucleotide sequenced (3).
We report here the isolation, cloning
and structural analysis of complementary DNA (cDNA) encoding turkey GH.
The
cloning of a GH cDNA from a third avian species adds to the existing database
*To whom correspondence should be addressed.
967
FAX 216-263-3675.
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
present for this biologically important molecule and permits the comparison of its amino acid and nucleotide sequences with those from a number of other mammalian and non-mammalian vertebrate species. With the potential of expressing large amounts of recombinantly derived turkey GH using eukaryotic expression vector systems (4,5) it should be possible to learn more about how GH regulates growth, metabolism and mineral balance in the turkey.
Furthermore, with the use of the cloned turkey GH cDNA
and genomic sequences (when they become available) it should be possible to learn how the synthesis of GH is regulated by hormones which enhance or inhibit GH gene transcription.
MATERIALS AND METHODS Isolation and culturing of pituitaries The anterior pituitaries were removed from 40 eight-week-old turkeys at the OARDC turkey research facilities. Organs were placed into sterile medium 199 plus antibiotics on ice. Extraneous tissue was removed and the pituitaries were minced and placed in static culture in fresh 199 medium plus antibiotics and i0 nM chicken gonadotrophin releasing hormone i (GnRH) for i hr at 41 ° C. (It has been reported that GnRH causes increased levels of GH secretion in goldfish (6)). Following, treatment glands were snap-frozen in liquid nitrogen. RNA isolation and eDNA library construction Total cellular RNA was isolated using the guanidinium isothiocyanate method: (7). Poly A containing mRNA was isolated by oligo dT cellulose chromatography as previously described (8). Turkey pituitary RNA that was double passed over oligo dT cellulose was used to prepare double-strand eDNA using the protocols described in the manufacturer's kit (Amersham, Arlington Heights, IL). The resulting methylated eDNA was blunted ended with the large Klenow fragment of DNA polymerase i, and EcoRl linkers were added. Following EcoRl digestion, the linkered eDNA was size fractionated by Sepharose 4B chromatography then ligated to EcoRl phosphatased lambda Zap II vector DNA (Stratagene Inc., La Jolla, CA)with recombinant constructs propagated in XL-I Blue E.coli (Stratagene Inc., La Jolla. CA). The primary library was titered and stored over chloroform. The IXI05 member library was 88% recombinant as determined by the chromogenic indicator X gal (9). eDNA cloning and sequencing strategy Approximately 50,000 plaques of the amplified turkey pituitary eDNA library were screened as previously described (i0). Duplicate nitrocellulose lifts were prepared as described (ii) then prehybridized (16 hrs) and hybridized (16-24 hrs) in non-fat dry milk/formamide solution (12). Filters were probed with a gel purified chicken growth hormone 807 bp eDNA insert (2) that was 32p labeled using the random primed oligonucleotide procedure (13). Several positive clones were selected for subsequent rounds of screening and four positive clones were plaque purified. Bluescript plasmids containing turkey GH cDNA inserts were excised from the lambda Zap II phagemid vector with helper virus (Stratagene Inc., La Joll~, CA). DNA from the four plasmids was Eco____Rldigested, separated by agazose gel electrophoresis, ethidium bromide stained, and Southern transferred to a nylon membrane (9). The filter was hybridized to 32p labeled chicken GH insert as above. All four plasmids contained inserts that hybridized to the probe. Clones i and 4 contained the two largest inserts (approximately 800 bp) and were selected for subsequent sequence analysis using the dideoxy chain termination method (14), primers KS and SK specific for the Bluescript vector (Stratagene Inc., La Jolla, CA) and the modified T7 polymerase, Sequenase (U.S. Biochemical, Cleveland, OH). Sequence data were compiled and analyzed using the MicroGenie Program (Version 6.0; Beckman Instruments, Palo Alto, CA).
968
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Northern blot analysis Forty pituitaries were removed from nine-week-old turkeys and placed in DMEM medium on ice. Pituitaries were separated into four equal groups and placed into culture dishes containing i0 ml DMEM plus antibiotics and incubated for 1 hr at 41 ° C with the following: I) medium alone, (control); 2) 1 n M h u m a n growth hormone releasing hormone (GRF, Peninsula Laboratories, Belmont, CA); 3) I0 nM somatostatin (SRIF, Sigma Chemical Co., St. Louis, MO); 4) I0 nM thyroid releasing hormone (TRH, Sigma Chemical Co., St. Louis, Mo). After incubation, the glands were snap-frozen in liquid nitrogen. Turkey brain and liver tissue were used as negative control tissue. Total cellular RNA was extracted from the tissues (approximately 150 mg) using the protocols previously described (7) then phenol extracted and ethanol precipitated. Ten micrograms of total cellular RNA from each of the samples was denatured in gel-running buffer (9) at 55 ° C for 15 minutes, fractionated by formaldehyde agarose gel electrophoresis and transferred onto a Zeta-probe nylon membrane (Bio-Rad, Richmond, CA) as described (9). The filter was baked as above, then prehybridized for 6-8 hrs at 42 o C in a diethylpyrocarbonate treated solution of non-fat dry milk that contained 509 formamide plus 19 SDS (12) and probed with the 32p labeled chicken GH eDNA insert (6-7XI0 6 dpm/ml). After overnight hybridization at 42 o C, the filter was washed in 2X SSC (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0) plus 0.19 SDS at 55 ° C, then 0.2X SSC plus 0.19 SDS at 60 ° C, and finally at 0.02X SSC plus 0.19 SDS at 65 ° C (45 minutes for each wash). The filter was exposed to Kodak XAR film for 3 hrs at -80 ° C. The level of probe hybridization to specific mRNA bands was quantified from autoradiographs using a scanning densitometer (E-C Apparatus, St. Petersberg, FL).
RESULTS Identification of cDNA coding for turkey growth hormone
A turkey pituitary
cDNA library was screened for GH using a purified heterologous 32p labeled chicken GH cDNA insert as probe.
Four plaque purified clones remained
positive after several rounds of screening.
Southern blot analysis of the
four recombinant plasmid cDNAs showed that all four eDNA inserts hybridized intensely to the probe.
The largest of the clones (i and 4) were selected for
nucleotide sequence analysis.
Figure 1 shows the composite nucleotide
sequence of the two different turkey GH eDNA clones (i and 4).
The turkey GH
nucleotide sequence contains a unique BamHl site that bisects the clones so that deletion of the EcoRl - BamHl fragment could produce sequence information from the internal portions of each clone.
Clone 1 lacked 7 bp (CAAAGGA) at
the 5 t end of the sequence shown in Figure 1 while it was determined that clone 4 missed the last two bp at the 3' end of the sequence (TG) as well as the poly A containing region.
Otherwise these two different clones were found
to be identical when overlapping sequences were compared.
In addition to the
sequence for the GH coding region, turkey GH clone 1 contains 41 bp of the 5'untranslated region (UTR), 92 bp of 3t-UTR and a 22 bp poly A tract while clone 4 contains 48 bp of 5' UTR, 90 bp of 3'-UTR and no poly A tract. poly A signal sequence AATAAA resides 14 bp from the poly A tract.
The
The turkey
GH eDNA clones 1 and 4 encode a 25 amino acid leader polypeptide and a 191 amino acid mature protein.
The deduced relative molecular weight (Mr) of the
mature protein is 22,272 daltons.
969
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-24 C•AAGGA•AGCTCACATTT•AACATC•TCCTGCATTTTCTCATCTTTCTGATTTATT•T•ATTGAACAAGGGAGAAAGATC ATG GAT M
D
88
-20 -i0 -i i TGC TAC AGG AAG TAT GCA GCT GTC ACT TTG ACC ATT TTG TCT GTG TTT CTG CAT CTT CTT CAT ACT TTC C Y R K Y A A V T L T I L S V F L H L L H T F
157
I0 20 CCA GAT GGA GAA TTT CTC ATG CAG GGT TGT CCA GAG TGC AAG CTA GGG GAG AAC AGG TTC TTT TCA AAA P
D
G
E
F
L
M
Q
G
C
P
E
C
K
L
G
E
N
R
F
F
S
K
30 40 CCA GGA GCC CCC ATT TAC CAG TGC ACT GGG TGC TGT TTC TCC CGG GCC TAT CCT ACT CCA ATG AGA TCC
226
P
G
A
P
I
Y
Q
C
T
G
C
C
F
S
R
A
Y
P
T
P
M
R
S
50 60 70 AAG AAG ACC ATG CTT GTT CCA AAG AAC ATT ACA TCG GAA GCA ACG TGC TGT GTA GCA AAG GCT TTC ACC
295
K
K
T
M
L
V
P
K
N
I
T
S
E
A
T
C
C
V
A
K
F
T
90
80
364
A
AAG ATT ACC CTT AAG GAC AAT GTG AAG ATA GAG AAC CAC ACA GAG TGT CAC TGC AGT ACC TGC TAC TAT K
I
T
L
K
D
N
V
K
I
E
N
H
T
D
C
H
C
S
T
C
Y
Y
96 CAT AAA TCT TAA AGCCTGTCCCTTTGCTAATGATCAAGAACAACGGTGAATGAAATATTTGTTGTTCAGCTTTTACAGCACCGCTGT
433
H
K
S
END
520
GTATAAT•TTGTGTTTT•TGGTCAAGACACCGAGTAGACTTTTGAATGAGATGGATGG•TGTTTTATTTCCTCTTTG•TT•TTCATGCATT
611
TAAGTAAGTTTAACTATTTCCATTAGGGATTAGATGTAGCCCTTGCATGACAACCATAAG•TTGATCTGTTTTTAAAATAAACTGCTAGAT
702
AAATTGTC [POLY A (45)]
Figure I.
The
Nucleotide sequence and corresponding amino acid coding sequence of turkey GH. The amino acids for the signal polypeptide are indicated by negative numbers while those for the mature GH protein are indicated by positive numbers.
size,
transcript Results
was
regulation
indicate
hybridized
specificity
that a s i n g l e p r o c e s s e d
to the
of p i t u i t a r i e s
and tissue
determined by northern blot
t u r k e y GH p r o b e
transcription
(lane 2) w h i l e
down regulate
t u r k e y GH m R N A r e s p e c t i v e l y
brain
(lane
5) a n d l i v e r
c D N A as e x p e c t e d
transcript
for c o n t r o l
with GRF caused a greater
of the
analysis
t h a n 20 f o l d
T R H a n d SRIF a p p e a r e d (lanes
that the p r o b e was
in F i g u r e
of a p p r o x i m a t e l y
pituitaries
induction
3 and 4).
Treatment
of t u r k e y GH
slightly
up a n d
RNA from turkey
to the l a b e l e d
tissue
2.
800 bp
(lane I).
to o n l y
(lane 6) d i d n o t h y b r i d i z e
indicating
t u r k e y GH m R N A
as s h o w n
turkey
GH
specific.
DISCUSSION Two d i f f e r e n t been
isolated
near
sequence
has b e e n
compared
to s e q u e n c e
high
degree
when
determined. data
When
growth hormone
The
compared
the c o d i n g
observed
t u r k e y GH also to the m a m m a l i a n
complementary
gland cDNA library region
for GH f r o m o t h e r a v i a n
of s e q u e n c e h o m o l o g y
respectively). 78%)
full-length
from a turkey pituitary
shares
of the t u r k e y GH is
species
(2,3)
there
(96% a n d 93% for c h i c k e n considerable
species human
970
DNAs have
and their n u c l e o t i d e
sequence
(15), b o v i n e
is a
and duck
homology (16),
rat
(68%(17),
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
2
3
4
800 bp -
Figure 2.
and mouse
Regulation of turkey GH transcription was performed by northern blot analysis. Total cellular RNA was prepared from nine week-old turkey pituitaries that received either no in vitro treatment (lane I); or I nM human GRF (lane 2); i0 nM SRIF (lane 3); i0 nM TRH (lane 4). Total RNA was also prepared from turkey liver (lane 5) and brain (lane 6) to serve as negative controls. Samples were fractionated by formamide gel electrophoresis, transferred to a nylon membrane, and hybridized to a 32p labeled turkey GH cDNA. The membrane was washed successively in 2X, 0.2X and 0.02X SSC at 55-65C, prepared for autoradiography and exposed overnight to Kodak XAR-5 film.
(18), but drops to 58~ homology when compared to salmon GH (19).
The nucleotide
sequence
chicken sequence
for the leader polypeptide
(97~ homology)
even less for the mammalian
species examined
82~) but only 46-54~ for the mammalian
(50~-58~).
species compared and 33~ for salmon GH.
of the chicken and duck (61~-78~)
and mammals examined
of the turkey GH cDNA agrees with other avian species but differs
contain additional There appears observed
The 5'-untranslated
homology with Chicken and duck (78-
the 3~-UTR of the turkey GH shares even less homology
(15,16,17,18)
(38~-42~).
from the salmon species
3F-UTR sequences
perhaps
~20 K variant'
important
The length
(19,20) which
for mRNA stability.
in the turkey GH cDNA as
of human GH.
thought to be the result of an alternative
than the 5'-UTR
(2,3) and mammalian
to be no smaller splicing variant
in the so-called
to the
but is less conserved for the duck (81~) and
region of turkey GH shares considerable
Overall,
is almost identical
The human variant
splicing of the pre-mRNA
is
transcript
(21). Figure 3 compares
the leader polypeptide
GH with several other species. 96~ homology mammalian
to chicken
species
is very homologous
The 25 amino acid signal polypeptide
shares
(2) and 83~ to duck (3), but only 24-32~ with the
that were compared to chicken
mammalian GH proteins
and mature protein for turkey
(15,16,17,18).
(96~) and duck (95~),
(15,16,17,18,22,23,24)
971
The turkey GH apoprotein 75-80~ homologous
while 69~ homologous
to
to the blue
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
-24
-20
-i0
Me••As••Cy•-Tyr•Arg-Lys•Tyr•A•a-A•a••a••Thr-Leu•Thr-I•e-Leu•Ser-Va•-Phe-•eu-His-Leu-Leu-His•Thr
chicken bovine human mouse
-
rat
Tyr Tyr Tyr
.
.
. . .
. . .
. . .
. . X X
. . .
. . .
. . .
. . .
. -
. . . Val-Thr Val-Met Val-Mec X X
.
. . -
-
Leu . . . Met Met-Val X X
.
.
Ile Val lie
-
-
Set Ser Set Set X
X
Ar K X
. X
X
X
X
Ile X
X
X
X
X
X
lie X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
equine
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
1
10
20
Iq~e~T~m~y~1u~Phe~Leu~t~e~n-G1y-C~s~Pr~-G1u~Cys~Lym-Leu-~y~u~Asn~Arg-I~he~Fbe~Ser-Lys .
.
.
human
Ala
-
mouse
Leu
-
rat porcine
Leu X
X
ovine
.
equine
.
.
-
Val
X
X
-
Asp
-
X
Asp-Leu-Ile-ne X X Thr
X
.
.
ASh Gln . . .
.
Thr
.
X
X
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. . Asp
-
.
.
Asp
.
.
.
. .
Thr . Thr-Thr
.
-
ne-ne
-
.
.
.
.
.
.
Thr
. .
chicken bovine
l'ro-Cly-AlaAsp .
human mouse
. . . . Leu . .
. .
.
.
rat porcine
Leu Leu
. .
. .
. .
ovlne
-
equine
Leu
. .
.
.
.
Gin .
.
.
Asp
.
-
.
.
VaI
.
Leu . . . . .
.
-
-
. . .
.
. .
.
Met Met
. .
Met
.
-Cys. .
Phe-Ser . .
. .
. .
. .
. .
chicken
. . .
rat porcine ovine equine
.
-Leu-Val-
.
.
-
Pro
Lys
-
Lys-Tyr
-
Lys Lys
-
Lys-Tyr Lys-Tyr
-
Lys Art
-
Lys -Tyr Lys-Tyr
-
-Ar&-Ala-Tyr-Plco-1[hr. . . . .
. .
. .
. .
. .
. .
chicken
.
. .
.
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
A1a Ala
.
.
.
.
.
.
.
.
.
.
.
Ala
Lys
.
.
.
.
.
.
.
.
.
.
.
Ala
.
.
.
.
.
.
.
Gln . .
.
.
Val . . .
.
.
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
. -
Val-Met-Giy-Gly-Phe -
porcine ovine
Val -Met -Gly Val-Met-Gly
-
equine
Val-Met-Gly
Figure 3.
-
- Pal Ala-Arg-Vai Ala-Arg-Val
Ala-Arg-Val Arg-Vai lie
-
Leu
Art
-Cya - Cym=Val-kla-Lys
.
.
Set . .
. .
.
. .
.
-Ala-
Phe -l~,r .Lyz- Ile
.
.
.
Ala
.
. .
. .
.
Set -Tyr -Asn-Arg.Val Ala
.
.
.
. .
. .
. .
. .
. .
. .
. .
.
.
.
.
Ala Aia
.
.
.
.
.
.
.
.
.
.
.
Ala
.
Set
.
Set
.
.
.
.
.
.
-
-
.
-
lle-Arg-Val
90
. . .
96
-Thr -Asp -Cys -His -Cys- Set -Thr- Cys .Tyr. Tyr .His .Lys. Set
. . . . . . . . .
.
Glu
.
.
Ala Glu . .
. . .
. .
.
.
. . .
. . .
. .
. .
. .
. .
Olu Glu
. .
. .
.
.
.
.
Gin
-
Tyr
. .
.
.
. . .
. . .
. . .
. . .
. . . .
. . .
. . . .
. . .
. . . .
. .
. . .
. . .
. . .
.
. .
. . His
-
Ile
The primary amino acid structure of turkey GH compared to other avian, mammalian and non-mammalian GH sequences. The leader sequences include amino acids -95 to -I and the mature protein include amino acids 1 to 191. Dashes indicate the same amino acids as for turkey GH. Lack of a comparable amino acid is shown by X. Comparisons of known amino acid sequences are for chicken (2), duck (3), bovine (16), rat (17), mouse (18), equine (22), human (15), and salmon (19).
(25) but only 55% homologous (26,27,28).
Surprisingly,
(89%) to the sea turtle
(29).
to salmon (19) and 40% for other fish turkey GH shows a high degree of homology
The four cysteine
181, and 189) which are important to structural same in all species
that were compared.
that are invariant regardless
(positions
115-120;
82-87;
evolutionarily
(positions
of the species compared
153-162) which suggest that these regions have been
site at the carboxyl
Asn-X-Asn)
binding or
functions.
When comparing avian GH proteins
there appears
to be no putative
termini of the turkey GH protein
as is found for chicken and duck (Asn-X-Thr).
of interest to utilize
53, 164,
folding were found to be the
conserved and might be important for ligand/receptor
specific biological
glyeosylation
residues
Figure 3 shows that there are regions
of block homology
may possess
Lys
.
Thr - Leu- Lys -Asp -ASh- Val -Lys -lle-Glu-Asn-Hls Val-Met-Gly Arg-Val . . . . Val-Met-Gly Val -Met-Gly
-
70
Pro - Lys -ASu- Ue -Thr - Set -Glu=Ala-Thr
.
.
.
Phe
Leu Ala
Met
. .
human mouse rat
Gln
pro -~let.~g.~r. Ala
. .
80 bovine
-
-
60
Lys -Thr -t~t
human mouse
Glm
Mec
50 ~vine
Lys-Tyr
~.0
Pro- I le -Tyr -ciu-Cys-Thz-Giy-Cys . . . . . Met: .
. .
-
-
.
.
.
Ly$
.
30
188-190;
-
. X
bovine
species
Gin
-
ovine
chicken
shark
-
porcine
X
.
lie Ile-Phe Ile
site-directed mutagenesis
techniques
(positions It would be
in order to change
turkey GH amino acid position 190 to either a Thr or Set to allow for posttranslational
glycosylation.
The production o f b o t h
972
recombinantly
derived
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
glycosylated as well as non-glyeosylated turkey GH in eukaryotic expression systems (4,5) would allow one to test the biological activity of the two GH isoforms. A number of peptide and steroid hormones acting alone and synergistically are known to regulate GH transcription and secretion.
The
northern blot data shown in Figure 2 (lane 2) reveals a large (greater than 20 fold by densitometry) and rapid (one hour) up-regulation of the steady-state levels of turkey GH mRNA suggesting that expression is at least in part regulated at the transcriptional level.
The observed increase in accumulated
levels of GH mRNA may be the result of increased transcription, message stability, or both.
A similar (albeit much lower) increase in mRNA levels
upon GRF induction has been observed in cultured bovine pituitary cells (30). SRIF administration has been shown to lower GH secretion in avian species (34,35,39) and the data in Figure 2 (lane 3) shows that SRIF marginally downregulates turkey GH transcription (.77 fold).
TRH has been reported to cause
increased GH secretion (31,32,33,34,35,36,37) in avian species and was shown in Figure 2 (lane 4) to cause a slight increase (1.85 fold) in turkey GH transcription as previously reported (38).
In addition to hypothalamic
peptides a number of peripheral hormones participate in the regulation of GH synthesis and secretion.
Insulin-like growth factor-i which mediates many of
the actions of GH, feeds back on the pituitary and hypothalamus to inhibit GH gene expression and secretion (36, 40).
Thyroid hormones, glucocorticoids and
retinoic acid act alone or in concert to enhance GH gene transcription (41,42,43,44).
The cloning of the turkey GH eDNA along with genomic sequences
in the near future should allow a better understanding of the complex hormonal control of GH gene expression.
ACKNOWLEDGMENTS The authors thank Dr. Mark Heiman, Eli Lilly and Company, Lilly Research Laboratories, Greenfield, IN, for the gift of human growth hormone releasing factor.
These sequence data will appear in the EMBL/Genbank/DDBJ Nucleotide
Sequence Data bases under the assession number M 33697.
Salaries and research
support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No. 300-90.
REFERENCES i.
2.
Souza, L.M., Boone, T.C., Murdock, D., Langley, K., Wypych, J., Fenton, D., Johnson, S., Lai, P.H., Everett, R., Hsu R.- Y., and Bosselman R. (1984) J. Exp. Zool. 232,465-473. Lamb, I.C., Galehouse, D.M., and Foster, D.N. (1988) Nuc. Acids Res. 16,9339.
973
Vol. 173, No. 3, 1990 3.
4. 5. 6. 7. 8.
9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19.
20.
21. 22.
23 24 25 26 27 28. 29. 30. 31. 32. 33. 34. 35. 36.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Chen, H.-T., Pan, F-M., and Chang, W-C. (1988) Biochim. Biophys. Acta 949,247-251. Leung, F.C., Jones, B., Steelman, S.L., Rosenblum, C.I., and Kopchick, J.J. (1986) Endocrinol. 119,1489-1496. Luckow, V.A., and Summers, M.D. (1988) Bio/Technol. 6,47-55. Marchant, T.A., Chang, J.P., Nahorniak, C.S., and Peter, R.E. (1989) Endocrinol. 124,2509-2518. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162,156-159. Aviv, H., and Leder, P., (1972) Proc. Natl. Acad. Sci., USA, 69,14081412. Maniatis, T., Fritsch, E.F., and Sambrook, J., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y Benton, W.D., and Davis, R. (1977) Science 196,180-182. Foster, D.N. (1987) Ohio Acad. Sci. 87,162-165. Johnson, D.A., Gautsch, J.W., Sportsman, J.R., and Elder, J.H. (1984) Gene. Anal. Techniques 1,3-8. Feinberg, A.P., and Vogelstein, B. (1983) Anal. Biochem. 132,6-13. Sanger, F., Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci., USA, 74,5463-5467. Martial, J.A.O., Hallewell, R.A., Baxter, J.D., and Goodman, H.M. (1979) Science 205,602-607. Miller, W.L., Martial, J.A., and Baxter, J.D., (1980) J. Biol. Chem. 255,7521-7525. Seeburg, P.H., Shine, J., Martial, J.A., Baxter, J.P., and Goodman, H.M. (1977) Nature 270,486-494. Linzer, D.I.H., and Talamantes, F. (1986) J. Biol. Chem. 260,9574-9579. Sekine, S., Mizukami, T., Nishi, T., Kuwona, Y., Saito, A., Sato, M., Itoh, S., and Kawauchi, H. (1985) Proc. Natl. Acad. Sci. USA 82,43064310. Nicoll, C.S., Steiny, S.S., King, D.S., Nishioka, R.S., Mayer, G.L., Eberhardt, N.L., Baxter, J.D., Yamanaka, M.K., Miller, J.A., Seilhamer, J.J., Schilling, J.W., and Johnson, L.K. (1987) Gen. Comp. Endocrinol. 68,387-399. DeNoto, F.M., Moore, D.D., and Goodman, H.M. (1981) Nuc. Acids Res. 9,3719-3730. Zakin, M.M., Pokus, E., Langton, A.A., Ferrara, P., Santome, J.A., Dellacha, J.M., and Paladini, A.C. (1976) Int. J. Pept. Protein Res. 8,435-444. Li, C.H., Chung, D., Lahm. H.-W., and Stein, S. (1986) Arch. Biochem. Biophys. 245,287-291. Li, C.H., Gordon, D., and Knorr, J. (1973) Arch. Biochem. Biophys. 156,493-508. Yamaguichi, K., Yasuda, A., Lewis, U.J., Yokoo, Y., and Kawauchi, H. (1989) Gen. Comp. Endocrinol. 73,252-259. Sato, N., Watanabe, K., Murata, K., Sakaguchi, M., Kariya, U., Kimura, S., Nonaka, M., and Kimura, A. (1988) Biochem. Biophys. Acta 949,35-42. Watahiki, M., Tanaka, M., Masuda, N,, Yamakawa, M., Yoneda, Y., and Nakashima, K. (1988) Gen. Comp. Endocrinol. 70, 401-40. Kawauchi, H., Moriyama, S., Yasuda, A., Yamaguchi, K., Shirahata, K., Kubota, J., and Hirano, T.K (1986) Arch. Biochem. Biophys. 244,542-552. Yasuda, A., Yamaguchi, K., Papkoff, H., Yokoo, Y., and Kawauchi H. (1989) Gen. Comp. Endocrinol. 73,242-251. Tanner, J.W., Davis, S.K., McArthur, N.H., French, J.T., and Welsh, T.H., Jr. (1990) J. Endocrinol. 125,109-115. Harvey, S. (1990) J. Endocrinol. 126,75-81. Harvey, S., Lea, R.W., and Ahene, C. (1990) J. Endoerinol. 126,83-88. Harvey, S. (1990) J. Endocrinol. 125,345-358. Scanes, C.G., and Lauterio T.J. (1984) J. Exp Zool. 232,443-452. Scanes, C.G. (1987) In Critical Reviews in Poultry Biology (R.R. Dietert, Ed.), Vol. i, pp. 51-105, CRC Press, Boca Raton, FL. Perez, F.M., Malamed, S., and Scanes, C.G. (1989) Gen. Comp. Endocrinol. 73,12-20.
974
Vol. 173, No. 3, 1990
37. 38. 39. 40. 41. 42. 43. 44.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Harvey, S., Foltzer-Jourdainne, C., Karmann, H., and Mialhe, P, (1988) Gen. Comp. Endocrinol. 70,374-381. Seo H. (1985) In The Pituitary Gland (H. Imura, Ed.) pp. 57-82, Raven Press, New York, NY. Harvey, S., and Scanes, C.G. (1987) Comp. Biochem. Physiol. 87A,315-318. Buonomo, F.C., Lauterio, T.J., Baile, C.A., and Daughaday, W.H. (1987) Gen. Comp. Endocrinol. 66,274-279. Seeburg, P. (1980) In Polypeptide Hormones (R.F. Beers, Jr., and E.G. Bassett, Ed.) pp. 19-31, Raven Press, New York, NY. Tushinski, R.J., Sussman, P.M., Yu, L.-Y., and Bancroft, F.C. (1977) Proc. Natl. Aead. Sci. USA 74,2357-2361. Nyborg, J.K., Nguyen, A.P., and Spindler, S.T. (1984) J. Biol. Chem. 259,12377-12381. Bedo, G., Santisteban P., and Aranda, A. (1989) Nature 339,231-234.
975
