Porcine Insulin-Like Growth Factor-I
(pIGF-l): Complementary
Deoxyribonucleic Acid Cloning and
Uterine Expression of Messenger
Ribonucleic Acid Encoding
Evolutionary Conserved IGF-I
Peptides*
Amir Tavakkol, Frank A. Simmen, and Rosalia C. M. Simmen
Department of Animal Science and Laboratories of
Molecular and Developmental Biology
Ohio Agricultural Research and Development Center
The Ohio State University
Wooster, Ohio 44691
INTRODUCTION
In order to facilitate studies of insulin-like growth
factor-l (IGF-I) expression during the pregnancyassociated development of uterus and mammary
gland in the pig model, we have isolated several
cDNA clones corresponding to porcine IGF-I (pIGFI) mRNA. Sequence analysis of two cDNA fragments
(sigf.2 and sigf.3) revealed an open reading frame
encoding in order a putative 25 amino acid (aa)
hydrophobic leader peptide, the mature (processed)
70 aa pIGF-l peptide and a 35 aa carboxy-terminal
extension (E) peptide. The deduced aa sequence of
the pIGF-l peptide is identical to human and bovine
IGF-I but differs from that of rat and mouse at three
and four residues, respectively. The sequences of
the amino- and carboxy-terminal IGF extension peptides are also highly conserved among these species. Northern analysis using sigf.3 as a probe revealed multiple IGF-I mRNAs (including species of
8000, 2300, and 1200 nucleotides in length) in uteri
of pregnant pigs. Highest levels of the uterine IGF-I
mRNAs were found at early pregnancy, when increased levels of immunoreactive tissue IGF-I were
also observed. Mammary levels of IGF-I mRNAs and
protein were considerably lower than that observed
for uterus at the same time period. Thus, uterine
production of IGF-I appears to be especially significant during early pregnancy in the pig when uterine
growth, elevated IGF-I in uterine fluids, and rapid
embryonic development are observed. (Molecular
Endocrinology 2: 674-681, 1988)
Insulin-like growth factor I (IGF-I) or somatomedin C is
a polypeptide growth factor of 70 amino acids (aa) in
length (mol wt, 7649) that is structurally related to
insulin (1). In vitro, IGF-I is a potent mitogen for a variety
of cell types including fibroblasts, myoblasts, chondrocytes, and erythroid precursor cells (1, 2). Binding of
IGF-I to specific cell membrane receptors elicits rapid
induction of protooncogene mRNA production and activation of ribosomal DNA promoters (3, 4), events
shown to be necessary to cellular mitogenesis.
IGF-I mRNAs are expressed in multiple tissues of
animals, with liver reportedly being the most significant
site of synthesis (5-9). Administration of GH to animals
leads to a rapid increase in steady state levels of IGF-I
mRNA in liver and other tissues (10-12). This is consistent with the proposed role of IGF-I as a major in
vivo mediator of GH action during postnatal growth. GH
binding promotes production of IGF-I mRNA and protein
in GH responsive tissues resulting in elevated levels of
circulating and tissue IGF-I. IGF-I acts in an endocrine,
paracrine, and/or autocrine fashion to promote cell
division, cell differentiation, and tissue morphogenesis.
Although a large body of information has emerged
concerning regulation of IGF-I production by GH (1,1012), less is known about (non-GH dependent) IGF-I
involvement and regulation during other tissue growth
processes. Of particular interest is the question of how
steroid hormones and peptides like IGF-I interact to
mediate the pregnancy-associated growth of reproductive tissues.
Several recent observations have suggested IGF-I as
a potentially important in vivo mediator of estrogen
0888-8809/88/0674-0681 $02.00/0
Molecular Endocrinology
Copyright © 1988 by The Endocrine Society
674
Characterization and Expression of Pig IGF-I mRNAs
action. Estrogen administration leads to rapid elevation
of uterine IGF-I mRNA levels in sexually immature rats
(13,14). Estrogen effects on granulosa cell proliferation
are also related in part to IGF-I production (15). Similarly, immunoreactive IGF-I molecules are found in the
developing mammary gland during pregnancy (Simmen,
F. A., R. C. M. Simmen, and G. Reinhart, submitted)
and are produced by cultured mammary carcinoma cells
(16), observations which implicate IGF-I as a possible
mediator of growth in this steroid responsive organ. As
an initial step to elucidate the physiological role of IGFI during normal growth of mammary and uterine tissues
in the pig, we have isolated and characterized several
porcine IGF-I (pIGF-l) cDNA clones. Using these cDNAs,
we further demonstrate the marked evolutionary conservation of IGF-I precursor proteins across mammalian
species and the differential expression of uterine IGF-I
mRNAs and protein at pregnancy.
675
Table 1. Pig Liver cDNA Clones Isolated by Hybridization
with a Murine IGF-I Probe
Bacteriophage
Isolate
Subclone
Designation
Fragment
Length (bp)
Southern
Hybridization
migf 1 -2
L6.1
L 18.1.1
L 18.2.2
sigf.1
sigf.2
sigf.3
sigf.4
sigf.3
459
620
580
900
bp, Base pairs.
sigf.1 to sigf.4, pig IGF-1 cDNA series. Bacteriophage and
plasmid DNAs were cleaved with EcoRI, fractionated in agarose gels, and subjected to Southern analysis with the indicated 32P-labeled cDNA fragments (see also Fig. 1). (+) and
(-) indicate hybridization or absence of hybridization, respectively.
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RESULTS
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Isolation and Analysis of cDNAs
Liver was chosen as the starting tissue for cDNA isolation since it is a significant source of IGF-I messenger
RNA in other species (5-9). A cDNA library constructed
in Xgt11 from adult pig liver mRNA was screened with
a 32P-labeled mouse IGF-I cDNA fragment (17). Three
bacteriophage clones which reproducibly exhibited hybridization under stringent conditions were identified
from an initial screening of approximately 500,000 recombinants (Table 1). DNA was then purified from each
of the three recombinant bacteriophages (L6.1,
L18.1.1, L18.2.2) and subjected to limited restrictionendonuclease analysis.
EcoRI digestion and agarose gel electrophoresis of
the cDNA clones yielded two insert fragments from
L18.2.2 and one from L18.1.1 and L6.1, respectively
(Table 1). Southern analysis using the 32P-labeled murine IGF-I probe resulted in strong hybridization to the
smaller (580 bp) fragment of L18.2.2 and the single
insert fragment of L18.1.1 (Fig. 1). Less hybridization
signal was observed to the 459 base pairs insert of
L6.1 whereas no specific hybridization was seen for the
larger fragment of L18.2.2 (Fig. 1). The three hybridizing
EcoRI fragments were subcloned into pBR322 for preparation of cDNA probes and into M13 vectors for DNA
sequence analysis (Table 1).
Sequencing of two of the subcloned cDNA fragments
(sigf.2 and sigf.3, Table 1) revealed an open reading
frame encoding a putative IGF-I protein precursor of
130 aa (Fig. 2). The sigf.2 and sigf.3 fragments were
colinear although the 5'-end of sigf.3 was extended by
48 nucleotides (nt) relative to the 5'-terminus of sigf.2
(Fig. 2). The 3'-ends of both cloned inserts mapped to
nearly the same position, except that the sigf.2 fragment also contained a long tract of poly dA:dT. sigf.3
lacks this region suggesting that this subclone is de-
459
Fig. 1. Southern Analysis of pIGF-l cDNAs
Purified bacteriophage DNAs (3 ^g each) were cleaved with
EcoRI, fractionated in an agarose gel, and subjected to Southern blot hybridization with 32P-migf 1 -2 fragment (see Materials
and Methods). DNA fragments displaying hybridization to the
murine IGF-I cDNA probe (17) were subcloned into appropriate
vectors (Table 1) and sequenced.
rived from an IGF-I mRNA which was polyadenylated
[poly(A)+] at a position further downstream. Accordingly, sigf.4 subclone of the original bacteriophage clone
(L18.2.2, Table 1) may correspond to this extended
region of 3'-untranslated region. The cDNA fragment
Vol 2 No. 8
MOL ENDO-1988
676
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TAGGAAGACCTTCCTGAAGAGTGAAGAATGACATGCCA
END
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CTGGCAGGATCCTTTGCTCTGCACMAGTTACCTGTTAMCACCAGMGACCTACCAAAAAMTAAGTTTGAAAACATT
TCAAAAGATGGGCATTCCCCCCAATGAAATACACAAGTAAACATTCCCGGAATTC
580
117
525
B
Fig. 2. Complementary DNA Sequences of sigf.2 and sigf.3 and Deduced aa Sequence of Pig IGF-IA Precursor Protein
The DNA sequences of sigf.2 and sigf.3 were determined in their entirety from multiple sequencing experiments. The DNA
sequence of sigf.3 and the corresponding amino acids are presented. Amino acids comprising the mature IGF-I peptide are
underlined. The first underlined residue (Gly) is taken as aa 1. The putative translational initiation codon (for met, -25) is boxed.
sigf.2 and sigf.3 differed at nt position 310 (C in sigf.3, T in sigf.2) which did not alter the aa usage, sigf.2 has a long poly-dA:dT
tract beginning at nt position 572 (of sigf.3) and both clones exhibit the variant poly(A)+ signal AATACA (nt 553-558 of sigf.3).
of sigf.1 did not cross-hybridize to the 32P-labeled sigf.3
probe on a Southern blot (Table 1) and DNA sequence
analysis of this clone revealed unique sequences with
no overlapping regions to sigf.2 or sigf.3.
Analysis of sigf.1 identified a segment of DNA homologous to a portion of the 5'-untranslated regions of
rat and mouse IGF-I mRNAs. However, the DNA flanking this conserved segment in sigf.1 is divergent from
the corresponding regions of the other IGF-I mRNAs
(not shown). It is likely that this cDNA clone was derived
from an incompletely processed or aberrant IGF-I
mRNA and therefore does not encode a functional IGFI protein.
Evolutionary Considerations
The 70 aa sequence of pig IGF-I deduced from cloned
cDNAs is identical to that of human and bovine IGF-I,
but differs from the rat and mouse protein at three and
four residues, respectively (Fig. 3A). Three of the four
differences noted with mouse IGF-I (migfi) are identical
to that observed with the rat protein. Comparison of
the IGF-IA carboxy-terminal (E) peptides (aa 71-105;
Fig. 3A) also revealed a high degree of sequence similarity. Only three aa differences were noted between
the corresponding human and pig peptides. Overall,
variation was found at only five of the 35 amino acid
positions when the four mammalian sequences were
compared. The IGF-I amino-terminal leader sequences
were also highly conserved. However, the predicted
initiator methionine residue at position - 2 2 of rat and
migfi (9, 11, 17) is replaced by a threonine in the pig
sequence. The putative initiator methionine for the pig
IGF-I precursor is at position - 2 5 which corresponds
to a methionine in the human protein (Fig. 3A). Comparison of residues - 2 5 to - 1 revealed variation at 5
aa positions.
Complementary DNA sequence comparisons for selected regions of human, rat, and pig IGF-I are shown
in Fig. 3B. Nucleotides corresponding to the leader
peptide [Met(-25) to Ala (-1)], mature IGF-I peptide, E
peptide (IGF-IA) and 3'-untranslated region (first 85 nt)
were 79%, 86.2%, 88%, and 63.5% identical, respectively. These correspond to sequences of exons 2, 3,
and 5 of the rat IGF-I gene (18). It is interesting that
90% identity was found for the portion of exon 5
encoding the last 18 amino acids of the E-peptide.
Uterine Expression of pIGF-l
The potential involvement of IGF-I in growth of female
reproductive tissues was initially examined by Northern
analysis of uterine RNAs from pregnant pigs using the
sigf.3 probe. This analysis revealed a major polyadenylated IGF-I mRNA of approximately 1200 nt with two
minor RNA species (8000 and 2300 nt, respectively)
677
Characterization and Expression of Pig IGF-I mRNAs
(-18)
Human
Rat
Hunan
Rat
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B
Fig. 3. Protein and cDNA Sequence Comparisons
A, Amino acid sequences for IGF-IA proteins of five mammalian species were compared with their differences indicated. The
sequences for the amino- and carboxy-terminal extension peptides of the bovine IGF-IA precursor protein are not available, so only
that corresponding to the processed 70 aa molecule is compared. Amino acid sequences are from Refs. 20 (human), 19 (bovine),
9,11,18 (rat), and 17 (mouse). B, Selected regions of cDNAs encoding pig, human, and rat IGF-I were compared and interspecies
variations were noted by asterisks. Nucleotide sequences are from Refs. 5, 20 (human), and 9,11,18 (rat).
superimposed upon a background of hybridization signal (Fig. 4). The band at 1200 nt appears diffuse,
possibly reflecting size heterogeneity within this class
of IGF-I mRNAs.
Steady state levels of IGF-I mRNAs were highest in
the uteri of early pregnant sows (day 12 of pregnancy)
and markedly declined during the later stages (days 60
and 90) (Fig. 4). These differences are significant since
equivalent amounts of poly(A)+ RNA (as monitored by
ethidium bromide staining) were electrophoresed in the
gels (data not shown). Northern analysis of poly(A)+
RNA from liver and mammary glands of pregnant sows
have so far failed to identify distinct IGF-I mRNAs, even
after prolonged autoradiographic exposure, although a
background of hybridization signal was apparent (Fig.
4). It is likely that this result reflects a lower level of
IGF-I mRNA in these tissues during pregnancy. In line
with this, our screening of 500,000 recombinants from
an adult pig liver cDNA library yielded only three IGF-I
clones, suggesting an IGF-I mRNA abundance level of
only approximately 0.0006% of total poly(A)+ RNA.
Quantification of tissue IGF-I was also carried out in
order to determine: 1) whether a decrease in levels of
IGF-I protein accompanied the observed changes in
uterine IGF-I mRNA abundance during pregnancy, and
2) whether IGF-I protein levels are lower in pig mammary glands at comparable developmental timepoints.
A panel of uterine and mammary tissues were extracted
with acetic acid to liberate IGF-I protein which was then
quantified by IGF-I specific RIA. The amounts of acidextracted immunoreactivity were expressed relative to
the total wet weight of starting tissue. As evident in Fig.
5, immunoreactive IGF-I was readily detected in both
the uterus and mammary gland at all stages of preg-
Vol 2 No. 8
MOL ENDO-1988
678
ceeded. THe actual amount of reduction in uterine
tissue IGF-I is probably more than that shown since the
contribution of blood-borne IGF-I to the total acid extracted pool would be expected to increase at later
pregnancy when extensive vascularization is present.
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DISCUSSION
2.3
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Fig. 4. Uterine Expression of IGF-I mRNAs in the Pregnant Pig
Poly(A)+ RNA was isolated from uteri of pregnant pigs or
from mammary glands of a pig at parturition and fractionated
in a 1.2% agarose-formaldehyde gel. After transfer to nitrocellulose, RNAs were hybridized with the 32P-labeled sigf.3
fragment. Lanes for each tissue contain 10 and 20 ^g poly(A)+
RNA, respectively. Panel A is an autoradiogram obtained from
shorter exposure time compared to that in panel B. Calculated
sizes in kilobases (Kb) of RNA species are based on relative
migration positions of 18 and 28 S ribosomal RNAs.
12
15
30
45
Day of Pregnancy
60
90
Fig. 5. Acid-Extractable, Immunoreactive IGF-I in Reproductive
Tissues of Pregnant Pigs
IGF-I protein levels were quantified by RIA after acid extraction of tissues (see Materials and Methods). Each developmental time point corresponds to a matched set of uterine
(shaded bars) and mammary (open bars) tissues from one
animal.
nancy. Interestingly, the IGF-I levels were higher in the
uterus of each animal and this tissue difference was
greatest at the early stages of pregnancy. A decline in
uterine IGF-I content was observed as pregnancy pro-
We report the complete amino acid sequence of pIGFI as deduced from sequence analysis of cloned complementary DNAs isolated by hybridization screening. This
extends to five the number of mammalian species for
which published IGF-I protein sequences are available
{i.e. human, bovine, rat, mouse, and porcine) (5, 9, 11,
17-19). Human, bovine, and pIGF-l peptides are identical and only differ from the rat and mouse protein at
3 and 4 aa, respectively. The aa sequences of the IGFI precursor proteins are also highly conserved. Two
variant precursor proteins have been described for
human, rat, and migfi (5, 9, 11, 17, 18, 20). These
molecules (designated IGF-I A and IGF-I B) differ in the
aa sequence of their respective carboxy-terminal (E)
peptides, a difference which is generated at the molecular level via alternative processing of IGF-I RNA transcripts. Two of our cDNAs (sigf.2 and sigf.3) correspond
to the IGF-IA mRNA form. None of our isolates encode
the IGF-IB precursor protein. This does not, however,
exclude the possible existence of IGF-IB mRNAs in pig
tissues since the number of cDNAs examined here were
limited.
The role of the IGF-I E-peptides remains for the most
part unknown. The remarkable conservation in length
and sequence of these peptides and their corresponding DNA sequences among mammals suggests a critical role or function, possibly dependent in part on the
class (IA, IB) of peptide expressed. It is possible that
secretory protein precursors must maintain a specific
chain length and sequence to enable their efficient
transport across the endoplasmic reticulum. The extension peptides may be important in this capacity for the
intracellular routing of newly synthesized IGF-I molecules. A second possibility is that these extension peptides are themselves hormone-like molecules with important biological functions. Alternatively, smaller peptides which might be generated from the E-peptides
may also be biologically active. The marked hydrophilicity of the pIGF-IA E-peptide contrasts with the alternating hydrophilic/hydrophobic nature of the mature
IGF-I molecule (data not shown).
The isolation of divergent 5'-untranslated regions for
IGF-I mRNAs (17, 21, 22) demonstrates that complex
splicing mechanisms are involved in IGF-I mRNA biosynthesis. This is consistent with the multiple hybridizing IGF-I mRNA species commonly observed in Northern blot analysis of other animal tissues (6, 8, 11, 1214,17), and may contribute in part to heterogeneity of
pig uterine IGF-I mRNAs.
We have demonstrated that uterine levels of IGF-I
protein and mRNA are relatively high in comparison to
679
Characterization and Expression of Pig IGF-I mRNAs
mammary gland levels during pregnancy in the pig.
Superimposed upon these tissue-specific variations are
the marked reductions in uterine IGF-I that occur as
pregnancy proceeds. The results of RIA and of our
limited Northern analyses suggest that IGF-I levels in
pig uterus are higher during the first weeks of pregnancy. The decline in IGF-I mRNA expression in uterus
as gestation proceeds may represent a major quantitative transition in IGF-I gene expression. Similarly, the
low level of IGF-I mRNA in the mammary gland and
liver may reflect a lower level of gene transcription or
perhaps a shortened half life for IGF-I mRNA molecules.
Early pregnancy in the pig is known to be an important period in the synchronous development and differentiation of the maternal uterus and early embryo (23,
24). Increases in uterine and endometrial wet weight
can be observed on a daily basis during this time.
Concurrently, pig embryos undergo a dramatic series
of morphological transitions along with underlying cell
division (23). This early embryonic growth is supported
in part by the presence of surrounding uterine luminal
fluid, which provides a source of nutrients (24). Porcine
uterine fluids also contain peptide growth factors that
can stimulate growth of embryo-derived cells in vitro
(25). One such factor, termed uterine luminal fluid mitogen, has been partially characterized and shown to
be distinct from IGF-I (26). IGF-I and uterine luminal fluid
mitogen are able to promote growth of pig uterine
fibroblastic cells in vitro (26). Consequently, growth
factors such as uterine IGF-I may serve as maternal
mediators of early embryogenesis by stimulating the
concerted growth of both embryonic and uterine cells.
Mechanisms underlying the local synthesis and secretion of growth factors to the uterine lumen remain
obscure.
IGF-I is also likely to be involved in mammogenesis
and lactogenesis of the pig since significant quantities
of this peptide are found in mammary secretions (Simmen, F. A., R. C. M. Simmen, and G. Reinhart, submitted) and mammary glands. However, the origins of this
IGF-I remain unclear. Other correlations exist for IGF-I
and augmented tissue development including growth
of bone (27), compensatory renal hypertrophy (28),
development of human placenta (29), and GH effects
on carcass characteristics of young pigs (30). The
availability of unique cDNA probes for pIGF-l mRNA will
enable further study of the mechanisms underlying IGFI expression in pig uterus and mammary gland and in
tissues of porcine fetuses and neonates.
MATERIALS AND METHODS
Library Screening
An adult pig liver cDNA library (1.7 x 106 members) (Clontech;
Palo Alto, CA) constructed in the Xgt11 expression vector (31)
was plated out (5 x 105 total pfus) on Escherichia coli strain
Y1088. Nitrocellulose filter replicates were prepared from the
agar plates as previously described (32) and prehybridized for
4-6 h at 65 C in a buffer containing 6x SSC (1 x SSC = 150
mM NaCI, 15 mM sodium citrate, pH 7.0), 5x Denhardt's
solution (0.1% ficoll, 0.1% BSA fraction V, 0.1% polyvinylpyrrolidone), 0.1% sodium dodecyl sulfate (SDS), and 30 /xQ/ml
yeast tRNA. Hybridization was carried out for 14-16 h at 65
C in the same buffer which also contained 10 mM EDTA and
the labeled probe (1-2 x 106 cpm/ml). After hybridization,
filters were taken through two successive washes in 2x SSC,
0.1% SDS for 10 min each, followed by two washes at 65 C;
first for 60 min in 2x SSC, 0.1% SDS, then for the same
period in 0.2x SSC, 0.1% SDS. Filters were air dried and
exposed to Kodak XAR-5 film for 14-16 h at - 7 0 C with
intensifying screens. Hybridization-positive plaques present in
duplicate filters were rescreened at lower plating density until
plaque purified. Four clones were isolated of which three
remained positive after Southern analysis.
Mouse IGF-I cDNA plasmid (migf1-2) (17) was digested with
EcoRI and the insert was gel purified from plasmid vector, and
labeled by nick translation or random-primer labeling with
[«32P]deoxyribonucleotide phosphates. Specific activities of
3-5 x 108 cpm/fig DNA were routinely obtained.
Preparation of Bacteriophage DNA
DNA from recombinant lambda bacteriophages was prepared
by the plate lysate method (32) with some modifications. In
brief, the phage stock (amplified to 3-5 x 1011 pfu/ml) was
used to infect E. coli strain Y1088. After bacterial lysis, plates
were overlayed with SM medium (32) for 14-16 h at 4 C.
Bacteriophage were pelleted and further purified by centrifugation onto a cushion of 1.5 g/ml cesium chloride in SM. DNA
was obtained by heating the phage particles to 65 C for 20
min in 0.2% SDS, 10 mM EDTA, followed by several phenolchloroform extractions. DNA was then precipitated with 2.5
vol ethanol, washed in 70% ethanol, dried, and dissolved in
an appropriate volume of Tris-EDTA buffer (32). An aliquot of
each sample was digested with EcoRI and electrophoresed
on a 1 % agarose slab gel.
Subcloning and Probe Purification
The EcoRI inserts of purified DNA from recombinant bacteriophages were ligated into EcoRI cut, dephosphorylated
pBR322. Plasmids were introduced into competent E. coli
DH5« cells (33), which were used for large scale plasmid
preparation (32). To purify cloned fragments, plasmid DNA
was digested with EcoRI, electrophoresed in 5% polyacrylamide gels, and the appropriate fragments eluted from acrylamide slices with 3 vol of 0.1 x SSC for 14-16 h at 4 C.
DNA Sequencing
Gel-purified DNA fragments were subcloned into M13 mp18
and mp19 vectors and sequenced using the Sanger dideoxy
chain termination method (34) and Sequenase (United States
Biochemical Co., Cleveland, OH). Sequence data were compiled and analyzed on an IBM XT computer (MicroGenie program, Beckman Instruments, Palo Alto, CA).
Materials
Animals
Restriction enzymes were purchased from Bethesda Research
Laboratories (Gaithersburg, MD), New England Biolabs (Beverly, MA), or Boehringer Mannheim (Indianapolis, IN). [«-32P]
dNTPs and [«-35S]dATP were obtained from ICN (Irvine, CA)
and Amersham Corp. (Arlington Heights, IL), respectively.
Uteri and mammary glands were removed from pregnant (days
6,12,15,30,45,60,90, and 115) gilts and immediately frozen
in liquid nitrogen and stored at - 7 0 C until use. Gestation in
the pig is typically 115 days. All animal protocols were con-
Vol 2 No. 8
MOL ENDO-1988
680
ducted under guidelines established by the American Veterinary Medical Association and the NIH. Animal use protocols
were approved by the Institutional Laboratory Animal Care
and Use Committee of The Ohio State University.
provided by State and Federal funds appropriated to the Ohio
Agricultural Research and Development Center. This is
OARDC Journal Article no. 93-88.
Isolation of poly(A)+ RNA
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Tissue RNA was prepared using the guanidine thiocyanate
procedure (32). Total RNA was pelleted by centrifugation of
the homogenates through a cushion of 5.7 M cesium chloride
for 24 h at 20 C. The poly(A)+ RNA fraction was obtained by
chromatography on an oligo(dT) cellulose column.
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Northern Analysis
Ten or 20 ^g poly(A)+ RNA was denatured in loading buffer
(24 ITIM HEPES, 6 IIIM sodium acetate, 1.2 mM EDTA, 50%
deionized formamide, 2.2 M formaldehyde) at 65 C for 20 min
and separated by electrophoresis in a 1.2% agarose-formaldehyde gel (32). Fractionated RNA was transferred to nitrocellulose paper as described (32). Blot prehybridization was
performed at 42 C for 4-6 h in 45% formamide, 6x SSC, 5x
Denhardt's solution.^0 ITIM sodium phosphate (pH 6.8), 0.5%
SDS, 2 [DM EDTA; 'and 30 /xg/ml yeast tRNA. Hybridization
conditions were identical to the above except that the Denhardt's solution was used at 1x, and the 32P-labeled sigf.3
probe added at 2-3 x 106 cpm/ml. Incubation was for 48 h,
at which point the filters were washed twice in 2x SSC, 0.1%
SDS for 20 min each, followed by a wash at 55 C for 60 min
in 0.2x SSC, 0.1% SDS. Hybridization signals were detected
by exposure of the filters to x-ray film at - 7 0 C.
IGF-I RIA
Acetic acid extracts of pig tissues were prepared according to
a modification of published procedures (28). Uterine and mammary tissues were homogenized in 1 M acetic acid (10 ml/g
tissue) and incubated for 2 h on ice. Homogenates were
clarified by low speed centrifugation and the supernatants
filtered through cheesecloth and saved. The pellets were extracted as above and the supernatants from each tissue
sample combined and lyophilized. The dried residue was suspended in 0.05 M Tris-HCI, pH 7.8 (2 ml/g starting tissue),
clarified by centrifugation, and used in RIA.
IGF-I RIA (35) was performed using rabbit antiserum
(UB286) raised to human IGF-I. Antiserum was obtained from
Drs. L. Underwood and J. J. Van Wyk through the NIDDK and
the National Hormone and Pituitary Program (University of
Maryland School of Medicine, Baltimore, MD). Recombinant
IGF-I (Thr59 analog, AMGEN Biologicals, Thousand Oaks, CA)
was used as tracer and standard. 125I-IGF-I was obtained from
Amersham Corp. (Arlington Heights, IL). Antigen-antibody
complexes were precipitated with goat anti-rabbit 7-globulin
and normal rabbit serum.
Acknowledgments
The authors would like to acknowledge Dr. Graeme Bell (University of Chicago, Chicago, IL) for providing the murine IGF-I
cDNA clone, Cindy Coy for assistance with DNA sequencing,
Linda Foster for plasmid DNA preparations, and Beverly Fisher
for secretarial assistance.
Received February 19,1988. Accepted April 25,1988.
Address requests for reprints to: Frank A. Simmen, Department of Animal Science, OARDC, Wooster, Ohio 44691.
* This work was funded in part by grants from the NICHHD
(New investigator Grant HD-22004) and The Ohio State University (to F.A.S.). Salaries and research support were also
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Fourth International Congress of Cell Biology
August 14-19, 1988
The Fourth International Congress of Cell Biology will be held on August 14-19,1988 at the
Montreal Convention Center, Montreal, Canada.
Abstracts may be accepted until the meeting date although they will not be included in the
program.
For further information, contact Congress Secretariat, Fourth International Congress of Cell
Biology, National Research Council of Canada, Ottawa, Canada K1A 0R6, Attn: K. Charbonneau, Congress Manager.