Download Nucleotide Sequence of the DNA Complementary to Avian (Chicken

Nucleotide Sequence of the DNA
Complementary to Avian (Chicken)
Preproparathyroid Hormone mRNA
and the Deduced Sequence of the
Hormone Precursor
John Russell and Louis M. Sherwood
Departments of Medicine (J.R., L.M.S.) and Biochemistry (L.M.S.)
Albert Einstein College of Medicine
Bronx, New York 10461
The nucleotide sequence of avian (chicken) preproPTH (prepro-PTH) mRNA was determined from a 2.3kilobase fragment of complementary chicken parathyroid DNA cloned in E. co/i MM 924. Northern blot
analysis of chicken parathyroid mRNA, using both
bovine and chicken cDNA probes, showed that the
mRNA (2.3 kilobases) for chicken hormone precursor was approximately 3 times the size of mRNA for
mammalian prepro-PTH. Cleavage of the cloned
DNA with restriction endonuclease Pst\ resulted in
three fragments, each of which was subjected to
sequence determination. The hormone sequence
deduced from the DNA showed that chicken preproPTH mRNA encoded a 119-amino acid precursor
which included a 25-amino acid signal sequence, a
six-residue prohormone peptide, and an 88-amino
acid hormone. The hormonal peptide was four residues longer than all known mammalian homologs
and included gene deletions and insertions. There
was significant homology of sequence in the biologically active 1-34 region with mammalian hormones,
but much less in the middle and carboxyl-terminal
regions. This is the first nonmammalian PTH sequence to be determined and should prove useful in
studying evolution of the gene as well as structurefunction relationships of the hormone. (Molecular
Endocrinology 3: 325-331, 1989)
end of the hormone molecule (2-4). At present, the
sequences of prepro-PTH from four different mammalian species (bovine, human, rat, and porcine) have been
determined (5-8). All four share a high degree of homology and consist of a precursor hormone molecule
of 115 amino acids, which includes a 25-member signal
sequence, a hexapeptide prohormone region, and an
84-residue hormone sequence. To investigate PTH
from nonmammalian species and to extend our studies
on its biosynthesis (9-11), we have cloned the cDNA
for chicken prepro-PTH mRNA. Northern blots of
chicken prepro-PTH mRNA and subsequent analysis of
the sequence of cloned cDNA demonstrated that it
shared considerable homology with mammalian preproPTH mRNA, but it also contained some striking differences. Chicken prepro-PTH mRNA was almost 3 times
larger than its mammalian counterparts (2.3 kilobases
compared with 0.8-0.9 kilobases), and it encoded a
larger hormone precursor containing 119 amino acids.
The additional length could be accounted for by the
hormone portion of the polypeptide, which contained
88 amino acids instead of 84. In this study we report
the sequence of several cDNA fragments which has
allowed us to deduce the primary structure of chicken
prepro-PTH as well as the structure of its mRNA.
RESULTS
INTRODUCTION
PTH is a major regulator of the serum calcium concentration. In conjunction with 1,25-dihydroxyvitamin D3,
the active metabolite of vitamin D, PTH acts on bone
and the kidney to mobilize calcium into the extracellular
fluid (1). The biological activity of PTH on its target
tissues has been shown to be present in a unique series
of amino acids from residues 1 -34 at the amino-terminal
0888-8809/89/0325-0331 $02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
In a wheat germ cell-free system, the poly(A+) RNA
fraction of chicken parathyroid glands translated a major protein component which migrated slightly ahead of
bovine prepro-PTH on sodium dodecyl sulfate (SDS)polyacrylamide gels (Fig. 1). This protein fraction could
be immunoprecipitated by antibodies to the aminoterminal portion of bovine PTH; the addition of a microsomal membrane preparation to the translation mix
converted it to a smaller protein, indicating the presence
of a signal sequence. When total chicken parathyroid
mRNA was analyzed by Northern blotting and hybridization with a cDNA probe containing the coding se325
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MOL ENDO-1989
326
kb
-4.4
43-
-2.37
-I.35
26-
-0.33
18-
1 2
B C
Fig. 1. Poly(A+) from Bovine (Lane B) and Chicken (Lane C)
Parathyroid Glands Was Translated in a Wheat Germ System
and Immunoprecipitated with Antibodies to the Amino-Terminal Portion of Bovine PTH
Translation products were separated on 15% SDS-polyacrylamide gels along with mol wt markers which included
ovalbumin and chymotrypsinogen, /Mactoglobulin, lysozyme,
bovine trypsin inhibitor, and insulin (a- and /3-chains).
quence for bovine PTH, a faint but distinct band was
observed midway between 28S and 18S ribosomal
RNA (Fig. 2). The size of the mRNA was calculated to
be about 2.3 kilobases. A cDNA library was generated
from the total poly(A+) fraction described, ligated into
the Pst\ site of PBr322, and used to transform E. coli
MM294 (12). Transformed colonies were screened with
a cDNA probe for bovine PTH mRNA. After screening
approximately 1000 colonies by in situ hybridization,
we selected 22 for further characterization. Since the
cross-hybridization between bovine and chicken PTH
sequences was weak, we used the additional technique
of hybridization selection (13) to eliminate any false
positives from the first screening step. Of the 22 positive clones from in situ hybridization, five (Pc-4, Pc-7,
Pc-9, Pc-17, and Pc-19) were also shown to be positive
by this procedure. Further characterization of the plasmids from these colonies showed that the one with the
largest cDNA insert produced three fragments when
cut with Pst\ (PC-4). The fragments contained a total of
Fig. 2. Northern Blot Analysis of Bovine and Chicken Parathyroid Gland Poly(A+) RNA
Approximately 5 ng chicken (lane 1) and 1 fig bovine poly(A+)
RNA (lane 2) were denatured in formaldehyde, separated on
a 1.5% agarose gel, and transferred to a nylon filter by
electroblotting. The filter was probed with nick-translated
cDNA for the coding region of bovine PTH mRNA, and autoradiography was performed overnight at - 8 0 C using Kodak
X-Omat AR film.
2.3 kilobases (see Fig. 3). When nick-translated and
hybridized individually to a Northern blot of total chicken
parathyroid mRNA, each one produced an intense band
in the region of 2.3 kilobases where earlier we had seen
cross-hybridization with the bovine cDNA probe. In
contrast to the bovine probe, however, the chicken
cDNA resulted in a signal that was approximately 100fold greater.
Sequence analysis (together with overlaps provided
by the other enzyme cleavages) showed that the smallest Pst\ fragment was at the 5' end, the intermediate
fragment in the middle, and the largest fragment at the
3' end (Fig. 4). An initiator codon (ATG) was found in
the middle of the smallest fragment, beginning an open
reading frame which could be read downstream from
this site for 357 bases, where it terminated in a stop
codon (TGA). The last 181 bases of open reading frame
were in the intermediate fragment. The longest fragment was placed at the 3' end and contained the
polyadenylation signal (AATAAA) and the poly(A) tail. A
second polyadenylation signal was identified in the intermediate fragment (see Fig. 4). The sequence of the
three fragments was confirmed by overlapping the fragments obtained with the other endonucleases (Haelll,
H/ndlll, and Taq\).
Sequence of cDNA to Chicken Prepro-PTH mRNA
327
500
I
HaeHI
I
Pc-4
Psll
HindlH
I
Toql
I
I
I
HaeEI TaqI
2000
I
1500
I
Hindi! Psll
I
1
Pc-9
1000
Hoelll
Toql
Haeffi
I
I
I
I
I
Toql
HaeUI
Toql
H
Fig. 3. Partial Restriction Map of the cDNA Insert from Pc-4 Showing Fragments Used to Determine the Sequence
Pc-9 is shown below which is identical to Pc-4, except that the 5' end is shortened by 156 bases.
SEQUENCE OF cDNA CORRESPONDING TO AVIAN PRE-PRO-PTH mRNA
100
GATTTAAGGGATCCACTAAACCAATTCAGTAGTAGTCTTTAAATATACTTGACATAAGACACAGCCArCTGCTGACArACCCCAACCAGAAAACTGTTAAGGACAATATCTGATAAAA
200
ATG ACT TCT ACA AAA AAT CTG GCC AAG GCC ATA GTG ATT TTA TAT GCT ATA TGT TTT TTT ACA AAC TCT GAT GGA AGA CCA ATG ATG
Met Thi Ser Thr Lys Asn Leu Ala Lys Ala lie Val lie Leu Tyr Ala le Cys Phe Phe Thr Asn Ser Asp Gly Atg Pro Met Met
AAG AGA TCG GTG AGT
Lys Arg Scr Val Ser
300
CTG CAG GAT GTG CAC
Leu Gin Asp Val His
GAG ATG CAA TTA ATG CAT AAC CTT GGA GAG CAT CGA CAC ACT GTG GAG AGA CAG GAC TGG CTT CAG ATG AAG
Glu Met Gin Leu Met His Asn Leu Gly Glu His Arg His Thr Val Glu Arg Gh Asp Trp Leu Gin Met Lys
AGT GCC CTT GAG GAT GCC AGG ACC CAG AGG CCT CGA AAC AAG GAG GAT ATT GTC CTG GGG GAG ATA AGA AAC
Ser Ala Leu Glu Asp Ala Arg Thr Gin Arg Pro Arg Asn Lys Glu Asp le Val Leu Gly Glu le Atg Asn
T
400
CGG AGG CTG CTC CCT GAG CAT TTG CGG GCA GCA GTG CAG AAG AAA TCC ATT GAC CTG GAC AAA GCT TAC ATG AAT GTA CTC TTT AAA
Arg Arg Leu Leu Pro Glu His Leu Atg Ala Ala Val Gin Lys Lys Set le Asp Leu Asp Lys Ala Tyr Met Asn Val Leu Ptie Lys
soo
ACT AAG CCA TGA TGA AAAGACCAAGAGCATTATAACTGCCAAGTAAGCACATGTTGTAGATCACTGACCAGTTAGGGCATTTTATTTATT
Thr Lys Pro MO
ATTTTTTTTTATTTAACTCAAACTATGATAAGGATTAAAGGCTCCATGCCAGACTGTAGCCCCACTGAGATGGGTATTTCACAACTAAATA
700
CTATATGGAGAGCATTTGTCTGTAATCTTTAGACCTACTAGTACTGTAAACTAACAACGTAATATAGGCATAACTGCATTAGTCCTAGGGT
1(KK>
ATAA^ATTGTGCATAAAAAGAAGAACAAGTTTTACATATACTGAAATGGAAGGGAGGTTTATTAACTTTCCCTCTTAATTATGAGCTGTCAC
1200
1
1300
GATAAAAACAAGACTTGTTTCAATTGTTATCATCTCTCCTTCAGTCAATAATCTATGAGTTTCTGTATATTGTGCTTAGGCCACATGGGTA
AGTGGCTCACATAAAATTACTCATCTTCACATGTGCACTTATACAGAAATTGGGATTTCAGTTTGTTAAAACCCTGAAATTACAACCATTA
AAATATAGAAATCAAAACCTGGGAACCATCAGTTAAAATATAAGCAGGATTCAGAAAGAATTTGACAGGAACATGGATGGGAGAAAATGAT
ibOO
GATTAATAATATAGAAAAGAAAGCAGCAAATATAAAATGATTTTGAATTGTATAGACAAGTAGTGCTTATGACCTCGACACCTTCTGAAT
GCCAACACATCCTGTGTGGTCCCATACCACTTCCTTGTTTGTTTGATGTCCTCGACACATCTTGGGAACGTAATGAAAAACTCATACATAATT
TACAAAATAAAGTGGACCAAAAGCTCTTGGACATTATTTTAAAGCATAAAAAAAAAAAAAAA
Fig. 4. Base Sequence of DNA Encoding Chicken Prepro-PTH mRNA
In the open reading frame (starting on line 2), the bases are separated as triplets, and the deduced amino acid sequence for the
hormone precursor of 119 amino acids is indicated. The open reading frame extends for 357 bases, followed by two TGA stop
codons. The Pst\ cleavage sites separating the three DNA fragments (Fig. 3) are indicated by arrows, and the two polyadenylation
signals are underlined.
Complete sequence analysis of the intermediate and
large fragments allowed us to place almost 1700 additional basepairs in sequence beginning at the 3' end of
the termination codon and ending at the poly(A) tail.
Figure 4 shows the base sequence for cDNA strand
corresponding to the sequence of chicken prepro-PTH
mRNA together with the deduced amino acid sequence
for the coding region.
A comparison of the amino acid sequence of chicken
prepro-PTH with those of bovine, human, and rat is
shown in Table 1. All four precursor hormones contained a 25-member signal peptide which terminates in
glycine. In this region the chicken sequence shared
44% homology with bovine, 48% with human, and 36%
with rat peptides. The six residue pro sequence of the
chicken precursor shared 50% homology with the rat
propeptide, but only 33% with both the bovine and
human sequences. The region that showed the most
conservation of homology with the mammalian hormones consisted of residues 1-34 of the hormone
sequence, which is the part of the molecule responsible
for hormonal activity (2, 3). In this region the chicken
sequence shared 59% homology with bovine, 65% with
human, and 65% with rat amino-terminal peptides.
Twenty residues in the 1 -34 region of all four hormone
sequences were identical, and all residues were encoded by the same triplet or had only a single substitution. At the remaining 14 positions, all but two resi-
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328
Table 1. Comparison of Chicken Prepro and 1-34 Sequences with Mammalian Counterparts
Conserved Amino Acid (%)
Prepeptide
Propeptide
PTH-(1-34)
Conserved Nucleotide (%)
Bovine
Human
Rat
Bovine
Human
Rat
11/25(44)
2/6 (33)
20/34 (59)
12/25(48)
2/6 (33)
22/34 (65)
9/25 (36)
3/6 (50)
22/34 (65)
47/75 (63)
11/18 (61)
70/102(69)
49/75 (65)
12/18 (67)
75/102(74)
42/75 (56)
13/18 (72)
65/102(64)
-20
-10
T[S]T[K]N L A[K]A I V I L YA
M[S]A[K]D M V[KJV H I V H L
-5
I C F F T N S D GR P N N K R S V S E
A I C F L A R S D GK S V K K R A V S E
IT
N H
M H
I|Q|F
20
30
[HIR H T
RK
[HJLSS
50
R P R N K E D I V L GITII
70
60
80
R N R R L L P E H L R A A V Q K K S I D L OKA
R P R K K E D N V L V|E|S HQ K S L G E A
Y N N
D K ADVD
88
V L
V L
I[K
Fig. 5. Comparison of Chicken and Bovine PTH Sequences with Regions of Homology Enclosed in Solid Black Lines
The two proposed deletion/insertion events are indicated.
dues (5 and 18) showed changes at two or three bases.
As was true for the mammalian hormones, the first 14
residues of the chicken hormone also showed a high
degree of homology (7 of 14) with the amino-terminus
of the PTH-related peptide associated with humoral
hypercalcemia of malignancy (14). Beyond the 1-34
region sequence homology between chicken and mammalian hormones was much less conserved. In fact, the
chicken hormone sequence was longer by four amino
acids, giving it a total length of 88 amino acids, compared with 84 for the mammalian sequences. The additional length and loss of homology could be explained
on the basis of gene deletions and insertions, which will
be discussed below (see Fig. 5).
The larger size of the chicken hormone was somewhat surprising, since chicken prepro-PTH migrated
faster than bovine prepro-PTH on SDS-polyacrylamide
gels (Fig. 1). The bovine precursor hormone migrated
with an apparent mol wt of 13,800, which was in good
agreement with its actual mol wt of 13,000. In contrast,
when SDS-gel electrophoresis was used to calculate
the mol wt of chicken prepro-PTH, it was determined
to be 11,250 compared with the actual mol wt of 13,920
based on the amino acid sequence. As yet, analysis of
the primary structure has yielded no obvious clues that
would explain the anomalous behavior of the peptide
on SDS-gel.
The 5' noncoding portion of chicken prepro-PTH
mRNA contained 115 bases and was slightly larger
than the 5' flanking sequences reported for mammalian
PTH mRNA. The 3' noncoding region was quite large
and consisted of almost 1700 basepairs, which was
1300 basepairs longer than the longest 3' noncoding
sequence reported for mammalian PTH mRNAs.
DISCUSSION
A 2.3-kilobase mRNA was identified in extracts of avian
(chicken) parathyroid glands which encoded a precursor
prepro-PTH molecule of 119 amino acids. The mRNA
was almost 3 times the size of its mammalian homologs
and encoded a precursor molecule four residues longer
than the mammalian precursors. In the deduced precursor peptide sequence, the signal and propeptides
were identical in size with the mammalian proteins, but
the hormonal peptide contained 88 residues rather than
84. Although the signal peptide of the chicken sequence
was identical in length with that in the mammalian
hormones, there was only moderate homology in this
region. The main regions of homology were in the
sequence around the cysteine at position 18 as well as
the glycine at position 25 which serves as the site of
cleavage of the leader sequence. The signal sequence
had a high degree of hydrophobicity, however, which is
characteristic of such sequences. In addition, the two
positive charges contributed by lysines at positions - 2 7
and - 2 3 were preserved in the chicken sequence,
which is in agreement with the concept that a net
positive charge in this region is necessary for secretory
function. The chicken hormone also contained a sixmember propeptide which ended in the dibasic sequence Lys-Arg, as is true for the mammalian homo-
329
Sequence of cDNA to Chicken Prepro-PTH mRNA
logs. Like mammalian prohormone sequences, it was
highly positively charged, with three instead of four
residues being basic. The dibasic carboxyl-terminal sequence serves as a recognition point for proteolytic
cleavage.
It was not surprising to find that the greatest conservation in sequence homology was in the amino terminal
1-34 portion of the hormones, since this region has
been shown to be responsible for the biological activity
of PTH (2, 3). Within this region, the amino-terminal
Ser-Val sequence is identical to that of human PTH,
and from previous studies has been shown to be required for biological activity (2, 6). In addition, two
domains have been delineated that bind the PTH receptor (15). These domains are located within residues 1 27 and 25-34, although the latter may be more important in binding activity. Although the 25-34 region of
the chicken PTH sequence contains many features in
common with the mammalian hormones, it lacks the
tribasic sequence from residues 25-27, which is
thought to be important for receptor binding (15). The
Arg-Lys sequence from 25-27 present in the mammalian hormones corresponds to Gln-Met-Lys in the
chicken sequence. In the region of the amino-terminal
peptide from residue 1-14 there was 7 1 % homology
(10 of 14 residues) between chicken and bovine peptides, with only 50% homology (10 of 20) between
residues 15 and 34. The amino acid sequence of the
tumoral peptide in humoral hypercalcemia of malignancy also shows striking similarity to mammalian PTH
in the 1-13 region (8 of 13 residues) (14). It will be of
interest to compare the biological activities of synthetic
chicken and mammalian hormones to determine
whether this change in structure affects its ability to
bind PTH receptors and produce specific biological
effects. In an initial study (16) we have compared the
biological activities of synthetic avian PTH(1-34) and
bovine PTH(1-34) in mammalian assays and showed a
marked relative decrease in the potency of the avian
peptide. A comparison of the amino acid hydropathy
(17) in this region showed that the change in amino
acid sequence resulted in a more hydrophobic environment around these residues in the chicken hormone,
which could alter its ability to bind receptors.
Beyond the 1-34 portion of the hormone, the homology was much less apparent. The additional length
of the chicken hormone and the difference in sequence
could be attributed to at least two deletion and insertion
events. The first deletion involves residues 32-44 of
the mammalian sequence, which is replaced by four
amino acids in the chicken sequence (see Fig. 5). This
occurs in the region where the mammalian hormone is
cleaved to produce the biologically active amino-terminal fragment (15). It is not known at this time whether
the chicken hormone undergoes similar proteolytic
processing, but if it does, the difference in sequence in
this region might imply that an enzyme(s) with a different
specificity may be involved. This first deletion/insertion
is followed by a region of strong homology from residues 37-53 of the chicken peptide, which corresponds
to residues 45-61 of the mammalian sequence. This is
followed by a second region of nonhomology resulting
from a deletion of residues 62-70 of the mammalian
sequence corresponding to an insertion of 22 amino
acids in the chicken sequence. A high degree of homology between the avian and mammalian sequences
is observed again in the COOH-terminal region corresponding to residues 76-88 in the chicken and residues
71-83 in the cow, human, and rat (see Fig. 5). The
chicken hormone sequence ends with two in-phase
termination codons (TGA TGA), which is similar, although not identical, to the human hormone sequence,
which terminates in TGA AAA TGA. Downstream from
the hormonal sequence was a 3' noncoding region of
almost 1700 basepairs, far longer than that for any of
the mammalian hormones. At least two polyadenylation
signals were present in the sequence (see Fig. 4), but
only one appears to be functional.
In these studies we have determined the structure of
the mRNA for avian prepro-PTH and the deduced hormone sequence. This is the first nonmammalian PTH
sequence to be determined, and it shows some interesting differences from mammalian homologs. In the
biologically active amino-terminal end there is significant
homology in the 1-14 region, but some critical changes
in the tribasic region (residues 25-27) thought to be
important for receptor binding. Our findings provide new
opportunities to study correlations of structure and
function and regulation of avian PTH gene expression.
MATERIALS AND METHODS
Preparation of Poly(A+) RNA
Newborn chicks were raised on a vitamin D3-deficient diet in
the dark for 5-6 weeks. The glands were harvested, and total
RNA prepared by the guanidinium isothiocyanate method (18).
The poly(A+) fraction was then isolated by chromatography on
oligo(dT) Sephadex (19). Approximately 1 ng poly(A+) RNA
was translated in 25 fi\ cell-free wheat germ extract using
labeled [35S]methionine (Amersham, Arlington Heights, IL).
Incorporation of [35S]methionine was typically 7- to 10-fold
compared with the blank reaction which contained no mRNA.
Translation products were characterized by electrophoresis
on 15% SDS-polyacrylamide gel electrophoresis.
Immunoprecipitation of Translation Products
Translation mixtures were diluted 10-fold with immunoprecipitation buffer (10 miui Tris, pH 7.4; 0.15 M NaCI; 10 ITIM EDTA;
0.5% Triton X-100; 0.5% sodium deoxycholate; and 3 mg
"gelatin/ml), and 63 /*g guinea pig antiserum to synthetic aminoterminal (residues 1-34) bovine PTH (Beckman, Fullerton, CA)
were added. The mixture was incubated on ice for 1 h, at
which time 10 n\ goat antiguinea pig 7-globulin (Arnel, New
York, NY) were added and incubated overnight at 4 C. The
mixture was spun at 15,000 x g for 10 min, and the pellet
was resuspended in SDS sample buffer containing 8 M urea,
boiled for 2 min, and subjected to electrophoresis on 15%
SDS-polyacrylamide gel electrophoresis.
Northern Blot Analysis
Approximately 2.5 ng total poly(A+) RNA were denatured by
incubation for 15 min at 55 C in buffer containing 50% form-
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MOL ENDO-1989
330
amide, 20 HIM (3-[N-Morpholine] propane-sulfonic acid) (pH
7.0), 6% formaldehyde, and 1 ITIM EDTA. The sample was
loaded onto 1.5% agarose gels and separated by electrophoresis for 4 h at 75 V. The RNA was transferred from the gel
to a 2 eta probe nylon membrane (Bio-Rad, Richmond, CA) in
10 nriM Tris acetate (pH 7.4) and 1 mM EDTA by electroblotting
at 250 mamps overnight. The dried membrane was hybridized
with a 650-basepair cDNA probe containing the entire coding
sequence for bovine prepro-PTH (12). The probe was labeled
by random primer extension (IBI) to a specific activity of 109
cpm/jtg DNA, and hybridization was carried out at 42 C
overnight in buffer containing 4x FSPE (1x FSPE = 0.15 M
NaCI, 10 mM Na2HPO4, and 1 mM EDTA, pH 7.4), 5x Denhardt's solution (1x Denhardt's = 0.01% Ficoll, 0.01% BSA,
and 0.01% polyvinyl pyrollidone), 0.5% SDS, and 0.05 mg/ml
salmon sperm DNA. After hybridization, the filters were
washed twice with 2x SSC (1 x SSC = 0.5 M NaCI and 0.015
M Na3 citrate, pH 7.0) at room temperature and then three
times in 0.1 x SSC at 55 C.
Preparation of cDNA and Amplification in E. coli
Single-stranded cDNA was prepared from the poly(A+) fraction
using reverse transcriptase (Bethesda Research Laboratories,
Gaithersburg, MD) by procedures described previously (12).
The reaction was terminated by extraction with phenol and
ethanol precipitation. The cDNA:mRNA hybrid was converted
to double-stranded DNA by the method of Gubler and Hoffman
(20), and double-stranded cDNA was poly d(C)-tailed and
annealed to Pst\ cut, poly d(G)-tailed PBr 322. The annealed
circular DNA was used to transform E. coli strain MM 294
(Clontech, Palo Alto, CA) by a modification of the method of
Hanahan (21). Colonies that contained plasmids with cDNA
inserts were ampicillin sensitive and tetracycline resistant, and
were selected using an AMP screen (Bethesda Research
Laboratories).
Identification of Chicken Prepro-PTH cDNA
Colonies were initially identified by filter hybridization (22) using
a radiolabeled cDNA probe for bovine prepro-PTH as described above. Weak hybridization between the bovine and
chicken PTH sequences necessitated the use of an additional
screening procedure, hybridization selection (13). Positive colonies were selected by their ability to hybridize selectively the
mRNA for chicken prepro-PTH, which could be eluted and
identified by translation in the wheat germ system. DNA inserts
were cut out by digestion with Pst\ and analyzed on 2%
agarose gels.
Sequence Analysis
cDNA fragments were subcloned into m13 (New England
Biolab, Beverly, MA) and sequenced by the dideoxy method
of Sanger (23). To provide overlapping sequences, additional
fragments were created by digestion with the restriction endonucleases H/ndlll, Haelll, and 7agl. All fragments were purified by gel electrophoresis and cloned into m13 vectors.
Ambiguities were resolved by sequencing both strands of DNA
and, in some instances, by substituting ITP for GTP to eliminate compression. To verify the sequence, two independent
clones were analyzed.
Acknowledgments
We wish to thank Ms. Deborah Lettieri for her excellent
technical assistance in this project and Dr. S. Hurwitz of the
Volcani Center, Israel for suppling the chicken parathyroid
glands.
Received October 19,1988. Accepted November 14,1988.
Address requests for reprints to: Louis M. Sherwood, Department of Medical and Scientific Affairs, Merck, Sharp and
Dohme International, P.O. Box 2000, Rahway, New Jersey
07075.
These studies were supported in part by Grants NIDDK
34822 and 28556 from the USPHS.
A preliminary account of the work was presented at the
10th Annual Meeting of the American Society of Bone and
Mineral Research, June 6,1988, New Orleans, LA.
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1,25-Dihydroxyvitamin D3 suppresses PTH secretion from
bovine parathyroid cells in tissue culture. Endocrinology
117:2114-2119
11. Russell J, Lettieri D, Sherwood LM 1986 Suppression by
1,25(OH2)D3 of transcription of the parathyroid hormone
gene. Endocrinology 119:2864-2866
12. Russell JR, Lettieri D, Sherwood LM 1983 Direct regulation by calcium of cytoplasmic mRNA coding for preproparathyroid hormone in isolated bovine parathyroid
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