From the
Human proteinase inhibitor 6 (PI-6) is a recently described
protein belonging to the serine proteinase inhibitor (serpin)
superfamily. Sequence similarity suggests that PI-6 most resembles the
ovalbumin (ov) serpins which include plasminogen activator inhibitor-2,
the squamous cell carcinoma antigen, monocyte/neutrophil elastase
inhibitor, and maspin. Although these proteins are associated with
carcinomas and inflammation, they appear to have diverse functions and
little is known of their physiological roles. In this study we have
characterized cDNA and genomic clones encoding mouse PI-6 in order to
analyze the localization, structure, and expression of the gene. The
reactive center residues (Arg-Cys) are conserved in the mouse molecule,
and recombinant mouse PI-6 was shown to bind thrombin, indicating that
it has similar inhibitory properties to its human counterpart. Using
reverse transcriptase-polymerase chain reaction assays on RNA isolated
from 15-day-old embryos and adult mice, we have shown that mouse PI-6
expression is developmentally regulated, and that, unlike human PI-6,
it is absent from the placenta. The mouse homologue of the human PI-6
gene has been designated Spi3 and was mapped to chromosome 13
between the Pl1 and ctla2
Serine proteinase inhibitors (serpins)
Recently, we and
others have identified a new serpin that closely resembles ov-serpins
in amino acid sequence and which also lacks a coventional signal
peptide(6, 7, 8) . This serpin was originally
designated the placental thrombin inhibitor (7) and cytoplasmic
antiproteinase(8) , but to avoid confusion the Genome Data Base
organization has recommended that it be known as proteinase inhibitor 6
(PI-6). Several facts suggest that, unlike most of the ov-serpins, PI-6
is an intracellular protein: it is not glycosylated (although it
possesses three potential sites for asparagine-linked carbohydrate); it
has never been detected in the medium of cultured cells; and it is
sensitive to oxidation(7) .
It is thought that closely
related serpins share common gene structures clustered at common loci
and that analysis of serpin gene configuration and position is a better
guide to evolutionary relatedness than amino acid similarity or
physiological function(9) . For example, a group of serpins
consisting of the protein C inhibitor, corticosteroid binding globulin,
In this study we have isolated and characterized a cDNA for mouse
PI-6, established the structure of the mouse gene, localized it to
chromosome 13, and examined its expression in the embryo and adult. We
find that it is expressed in most adult tissues and appears to be
developmentally regulated. It is almost identical in gene organization
with PAI-2 and ovalbumin, except that it lacks an intron present in
both genes and it does not co-localize with PAI-2. This suggests that
PI-6 does not belong to the ov-serpin branch of the serpin family.
To isolate the 5` end of the mouse PI-6 cDNA, anchored PCR (13) was employed. Briefly, mRNA prepared from BALB/c 3T3 cells
was used as a template for the synthesis of cDNA(14) . A
50-µl reverse transcription reaction was carried out at 42 °C
for 2 h and contained 1 µg of mRNA, 0.5 µg of a purified
oligonucleotide (PB-92; ), 400 µM dNTP, 10
units of RNasin (Promega), and 20 units of avian myeloblastosis virus
reverse transcriptase (Promega). The reaction was ethanol-precipitated
and resuspended in 50 µl of H
BALB/c 3T3 cells were incubated in 5%
CO
To isolate the 5`
end of a mouse PI-6 cDNA, an anchored PCR strategy was
used(13) . mRNA prepared from BALB/c 3T3 cells was used as a
template for cDNA synthesis primed by oligo(dT). An anchoring
oligonucleotide (blocked at the 3` end by addition of ddATP using
terminal deoxytransferase) was ligated to the cDNA using T4 RNA ligase,
and the cDNA was then used as a template for PCR using one primer
complementary to the anchoring oligonucleotide, and the other primer
antisense to a 5` region of the mouse PI-6 coding sequence. A resulting
330-bp product was verified by hybridization to a specific
oligonucleotide designed to a sequence upstream of the antisense
(PI-6-specific) primer. DNA sequence analysis confirmed that this
330-bp fragment contained the mouse PI-6 5`-untranslated region.
The
complete sequence of the mouse PI-6 cDNA was assembled from the longest
partial cDNA clone arising from the libraries and from the 5` PCR
product (see Fig. 1). Sequence data were derived by walking down
both strands using specific oligonucleotide primers (). A
1.2-kb full-length cDNA clone was then amplified from the BALB/c 3T3
cDNA by PCR using a proofreading polymerase and primers corresponding
to the 5` and 3` ends of the predicted mouse PI-6 mRNA. The resulting
product was cloned and entirely sequenced. There were no differences
between the 5` sequences of this full-length cDNA and the 330-bp
fragment, and both matched the corresponding genomic sequence,
suggesting that neither clone contained PCR-introduced mutations.
A PvuII restriction fragment length
polymorphism between M. musculus and M. spretus genomic DNA was detected by hybridization of a XbaI-HindIII probe consisting of part of intron A,
exon II, and part of intron B of the mouse PI-6 gene (see Fig. 3). Hybridization of this probe to a panel of 94 PvuII-digested DNAs made from backcross mice (BSS(18) )
localized the PI-6 gene to chromosome 13 (Fig. 4). Data in show the recombination frequencies for the nearest linkage
markers for the mouse PI-6 gene. No recombinants were found between the
PI-6 gene and the motif-primed PCR marker D13Bir8, which is
positioned approximately 20 centimorgans from the centromere on
chromosome 13(18) . On the map of chromosome 13, the PI-6 gene
falls between the placental lactogen 1 gene (3.2 centimorgans proximal)
and ctla2
To show that
these sequences constitute a functional promoter, a 680-bp fragment
(-650 to +31) was subcloned and placed in front of a
bacterial chloramphenicol acetyltransferase (CAT) gene in a plasmid
derived from pSVTf-CAT(17) . The subcloned fragment contains the
5`-flanking sequences and 31 bp of the 5`-untranslated region of the
structural gene (Fig. 1). The resulting plasmid (pPI-CAT) was
used to co-transfect BALB/c 3T3 cells along with a vector carrying a
G418 resistance gene. G418 resistant clones were pooled, and cell
extracts were assayed for CAT activity. Parallel experiments were
carried out with a positive control plasmid (pSVTf-CAT) and a negative
control plasmid (p2CAT). Assays for CAT activity on cell lysates showed
CAT production in cells containing pSVTf-CAT and pPI-CAT, but not in
cells containing p2CAT (Fig. 5). These results show that the
region immediately adjoining exon I of the PI-6 gene can function as a
promoter.
As shown in Fig. 6, many
tissues in the adult produce PI-6, with the highest levels observed in
brain and liver. In the embryo (Fig. 7), PI-6 production was
noted predominantly in kidney, lung, and yolk sac. Levels were low or
absent in the placenta, liver, heart, brain, and muscle. The different
patterns of expression between adult and embryo in organs such as liver
and brain suggest that PI-6 is developmentally regulated.
We have isolated and characterized cDNA and genomic clones
encoding the mouse homologue of human PI-6, which has been designated
serine proteinase inhibitor 3 (Spi3) by the International
Committee on Standardized Nomenclature for Mice.
Mouse and human
PI-6 show 76% similarity at the protein level and are most closely
related to the ov-serpins(2) . Of particular interest is the
conservation of the residues (Arg-Cys) at the predicted reactive
center, suggesting that the two molecules have similar inhibitory
properties. This is supported by our observation that, like human PI-6,
the mouse molecule interacts with thrombin. The conservation between
mouse and human PI-6 also extends to the unusually large number (for
serpins) of cysteine and methionine residues in the molecule,
particularly in the reactive center region. For human PI-6 we have
shown that this feature renders it sensitive to oxidation(7) ,
and this is also likely to be the case for the the mouse molecule.
Like the rest of the ov-serpins, both mouse and human PI-6 lack
discernible amino-terminal signal sequences. Although most of the
ov-serpins can be glycosylated and secreted using unconventional
signals, there is no evidence of glycosylation in native or recombinant
human PI-6(6, 7, 8) , and we have recently shown
that the molecule cannot exit the cell via the classical secretory
pathway and that artificially glycosylated PI-6 has no proteinase
inhibitory activity.
Analysis of mouse PI-6 gene
expression shows that PI-6 is present in many tissues of the adult and
embryo. Tissues not surveyed in our previous analysis of human PI-6
mRNA distribution include spleen, thymus, and testis, all of which
express PI-6. One obvious difference between the species is the absence
of PI-6 from the mouse placenta. There are two possibilities to explain
this: PI-6 is only expressed in the placenta at or near term (day
19-20 in the mouse) or PI-6 in the mouse yolk sac (a structure
not present in humans) fulfills a function provided by PI-6 in the
human placenta. (It is interesting to note that PAI-2 is also found in
the human but not the mouse placenta(5) .)
PI-6 levels
apparently alter in various organs during development. For example,
compared to adults, embryos express relatively low levels of PI-6 in
liver and brain and relatively high levels in the kidney. The finding
that PI-6 is present in embryonic stem cells suggests that the gene is
expressed soon after conception and may play a role in differentiation
and development. Further analysis of PI-6 expression in embryos of
varying ages and neonates is required to explore this possibility.
Changes in PI-6 levels during development is the first indication of
regulated expression of this gene. To date, PI-6 has been noted in a
wide variety of tissues and cultured cells, but, unlike
PAI-2(5) , alterations in levels in response to growth factors,
cytokines, or tumor promoters has not been observed. The 600-bp region
flanking the 5` end of the PI-6 structural gene contains elements of
the promoter, as shown by its ability to direct transcription of a
heterologous gene. Inspection of the sequence reveals a TATA box but no
CAAT box upstream of the transcriptional initiation point. However,
there is a GC-rich region near the TATA box which may function as an
SP1 binding site (22). There are no other discernible motifs for the
potential binding of transcription factors, except for an indirect
repeat centered at nucleotide -443, and a direct repeat centered
on nucleotide -192 which resembles a binding site for members of
the C/EBP family. C/EBP is found in tissues such as brain, liver, lung,
and skin and is thought to be involved in regulating the balance
between cell growth and differentiation(22) .
Soon after the
serpin protein family was recognized, interest focused on serpin gene
structure. Surprisingly for a family of proteins with such high
structural and domain homology, a considerable degree of variation in
both the numbers and positions of intron/exon boundaries was noted
(reviewed in Ref. 9). This led to two models for serpin gene evolution.
The first proposed an ancestral gene containing three introns from
which succeeding genes were generated by duplication and intron
insertion and deletion. In the second model, an ancestral gene
containing at least 22 introns, each marking the borders of distinct
domains in the protein, was proposed, from which other serpin genes
were generated by duplication and intron deletion(23) .
A
number of different serpin gene configurations have now been
identified, and there is mounting evidence that the serpin family may
be divided into subgroups on the basis of gene position and
organization, and that this is a better guide to evolutionary
relatedness than amino acid similarity or physiological function. For
example, the human protein C inhibitor, corticosteroid binding
globulin,
The
observation that human PAI-2 and chicken ovalbumin have identical gene
structures is consistent with this view and has been used to argue that
PAI-2 is the mammalian homologue of ovalbumin(11) . It can also
be predicted that the ov-serpins closely resembling ovalbumin and PAI-2
will have similar organizations and locations. Mouse PI-6 is the third
potential member of the ov-serpin group for which a gene configuration
and localization have been described. As shown in Fig. 8, the
structure of the PI-6 gene closely resembles those of PAI-2 and
ovalbumin and is distinctly different from other members of the larger
serpin family. However, PI-6 lacks an intron present in both ovalbumin
and PAI-2 (intron C) and is present on chromosome 13, in a region
syntenic with human chromosome 6. This would suggest that human PI-6 is
located on chromosome 6, and our recent results confirm
this(26) . On the other hand, PAI-2 is localized on human
chromosome 18 and mouse chromosome 1(27) , so PI-6 and PAI-2
cannot be part of the same gene cluster. On this basis we propose that
PI-6 is not a member of the ov-serpin group, and that it represents a
new serpin type that has recently diverged from the ov-serpins. It
remains to be seen whether the other ov-serpins (maspin,
monocyte/neutrophil elastase inhibitor, squamous cell carcinoma
antigen) resemble PAI-2 or PI-6 in gene organization and localization.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Lucy Rowe (The Jackson Laboratory, Maine) for
analysis and review of the gene mapping data, Dr. Marie Dziadek (Monash
University) for assistance with mouse embryo dissections, and Dr. Paul
Coughlin for discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
genes. It spans 20
kilobases, consists of 7 exons and 6 introns, and contains a TATA motif
24 nucleotides upstream of the transcriptional start site. A 680-base
pair DNA fragment containing this motif and 31 nucleotides of the
5`-untranslated region of the structural gene directed transcription of
a bacterial cat gene, demonstrating the presence of a
functional promoter. The PI-6 gene lacks an intron present in the
ovalbumin and PAI-2 genes; otherwise it is identical in terms of the
numbers, position, and phasing of the intron/exon boundaries. These
results suggest that PI-6 and the ov-serpin genes have diverged and do
not belong to the same subgroup.
(
)are a family of proteins that regulate the activity
of serine proteinases involved in extracellular processes such as
coagulation, fibrinolysis, complement fixation, and embryo
implantation(1) . A group of five proteins (ovalbumin, maspin,
squamous cell carcinoma antigen, plasminogen activator inhibitor 2
(PAI-2), and monocyte/neutrophil elastase inhibitor) make up a branch
of the serpin family known as the ovalbumin (ov) serpins(2) .
The physiological roles of these inhibitors are not understood, but it
is thought that they function in extracellular processes because
alterations in their production or release are associated with
tumorigenesis and inflammation. For example, loss of maspin correlates
with increased malignancy and metastasis of breast
carcinomas(3) ; increases in the levels and release of squamous
cell carcinoma antigen occur in squamous cell carcinomas(4) ;
and increased expression of PAI-2 occurs in monocytes during
inflammation(5) . The ov serpins are unusual because each lacks
an amino-terminal signal peptide normally required to initiate
secretion and glycosylation of the protein. Nevertheless, secretion and
glycosylation of most of the ov-serpins (ovalbumin, maspin, squamous
cell carcinoma antigen, PAI-2) does occur, mediated by nonconventional
signal sequences present elsewhere in the protein.
-1 proteinase inhibitor, and
-antichymotrypsin
have similar gene structures which are clustered on human chromosome
14(10) . In the case of the ov-serpins, it has been shown that
chicken ovalbumin and human PAI-2, although separated by millions of
years of evolution, have identical gene organizations in terms of the
numbers, positions, and phasing of the intron/exon
boundaries(11) . It can be predicted that PI-6 will exhibit a
similar gene organization if it is a member of the ov-serpin group.
General Methods
General recombinant DNA methods
were as described in Ref. 12. cDNA probes were labeled with
[-
P]dCTP (DuPont NEN) using the random
hexamer method (NEBlot
Kit, New England Biolabs).
Oligonucleotides were labeled with [
-
P]ATP
(DuPont NEN) as described(12) . Conventional DNA sequencing was
carried out using the Sequenase II system (Amersham). Cycle sequencing
was carried out using the CircumVent
Kit (New England
Biolabs). Oligonucleotide primers for PCR and sequencing were
synthesized by Bresatec (Australia).
Isolation and Sequencing of Partial Mouse PI-6
cDNAs
4 10
plaques of a mouse liver cDNA
library in
gt10 (Stratagene) were screened with a full-length cDNA
encoding human PI-6 (7). Filters were hybridized in 50% (v/v)
formamide, 6
SSC at 32 °C overnight, and washed in 2
SSC at 45 °C. Inserts from 3 positive clones were released
by EcoRI digestion and subcloned into Bluescript II
KS
(Stratagene) for sequence analysis. All contained
short (up to 600 bp) sequences homologous to the 3` end of human PI-6
cDNA. The longest fragment was then used to screen a similar number of
plaques from a second mouse liver cDNA library in
gt11. 10
positive clones were obtained, all containing approximately 1.2-kb
inserts. Restriction mapping showed that each insert contained 2
internal EcoRI sites. Each of the three fragments was
subcloned and sequenced. In addition, primers specific for
gt11
were used to amplify and subclone a complete copy of the insert.
Sequence analysis of each of the EcoRI fragments and the PCR
fragment confirmed that a mouse homologue of PI-6 had been isolated.
However, all of these clones lacked 5` sequences present in human PI-6.
O. A portion of the cDNA
was then ligated to the anchoring oligonucleotide (PB-40; )
that had been 5`-phosphorylated and blocked at the 3` end by incubation
with ddATP and terminal deoxytransferase as described(13) . The
20-µl ligation reaction contained 2 µl of the cDNA, 20 pmol of
the anchoring oligonucleotide, 25% (w/v) PEG 8000, and 20 units of T4
RNA ligase (New England Biolabs) and was incubated overnight at room
temperature. The following day, 64 µl of 0.5 M NaCl, 10
mM Tris, pH 8, 1 mM EDTA was added to terminate the
reaction. 2 µl of the terminated ligation was used as a template
for PCR. This PCR (performed in duplicate) used 40 pmol of the primers
PB-103 and PB-91 () and contained 50 mM Tris, pH
9.0, 50 mM KCl, 0.1% (v/v) Triton X-100, 1.5 mM MgCl
, 200 µM dNTPs, and 1 unit of TthI. 35 cycles of 95 °C for 90 s, 50 °C for 60 s, and
70 °C for 90 s were performed. The resulting 400-bp products were
subcloned into the plasmid pCRII (Invitrogen). Sequence analysis
confirmed the presence of sequences homologous to the 5` end of human
PI-6.
Construction of a Mouse PI-6 Expression Vector
A
PCR-based strategy was employed to obtain a full-length mouse PI-6
cDNA. mRNA from BALB/c 3T3 cells was reverse-transcribed as above
except that 0.5 µg of poly(dT) primer was used. A PCR containing
0.5 µl of the cDNA, 40 pmol of the primers PB101 and PB105 (), 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10
mM (NH)
SO
, 2 mM MgSO
, 0.1% (v/v) Triton X-100, 200 µM dNTPs, and 2 units of Vent
polymerase (New England
Biolabs) was performed. The reaction was cycled 35 times at 95 °C
for 90 s, 50 °C for 60 s, and 72 °C for 90 s. The resulting
1.2-kb product was cloned into the pCRII vector and sequenced
completely. The cDNA was released from the pCRII plasmid by digestion
with EcoRV and SpeI and was cloned into pSVTf (15) digested with SmaI and XbaI. Mouse PI-6
mRNA was synthesized in vitro from the resulting plasmid by T7
RNA polymerase and translated in a rabbit reticulocyte lysate system as
described(16) .
Analysis of Mouse PI-6 RNA Distribution by Reverse
Transcriptase-PCR
An adult mouse was dissected, and total RNA
was isolated from various organs as described(14) . RNA was also
isolated from the pooled organs of five 15-day-old embryos. Reverse
transcription using 10 µg of RNA was performed as described above.
To control for DNA contamination in the subsequent PCR, a parallel
reaction without avian myeloblastosis virus reverse transcriptase was
performed. A PCR using TthI polymerase was performed as
described above except that it contained 4 primers: 5 pmol of the
glyceraldehyde-3-phosphate dehydrogenase specific primers (PB-120 and
121; ), as well as 25 pmol of the mouse PI-6 primers PB101
and 105 (). 35 cycles were performed; 95 °C for 90 s,
50 °C for 60 s, and 70 °C for 90 s. The PCR products were
separated on 2% agarose gels, visualized by ethidium bromide staining,
and then transferred to nylon for Southern hybridization using a P-labeled oligonucleotide (PB92).
Isolation and Characterization of the Mouse PI-6
Gene
1 10
plaques from a 129/Sv mouse
genomic DNA library in
FIX II (Stratagene) were screened using the
full-length mouse PI-6 cDNA as a probe. Conditions were as described
above except that the hybridization was performed at 42 °C and the
final wash was in 0.5
SSC at 65 °C. DNA was prepared from
the positives using the Wizard
Lambda Preps DNA
Purification System (Promega). Intron/exon boundaries were identified
by sequencing the two clones directly using specific oligonucleotide
primers (). DNA from each clone was used as template in a
cycle sequencing reaction (CircumVent
; New England
Biolabs). The size of each intron was estimated by analysis of the size
of the products resulting from a PCR using flanking primers () in a TthI-based protocol (see above) .
Mouse PI-6 Promoter Analysis
The genomic clone
7 was digested with HindIII and a 6-kb fragment
containing the 5`-flanking region, exon I, intron A, exon II, and part
of intron B was subcloned into pUC118 (pUC118/7H3). A 700-bp HindIII-BglII fragment containing the 5`-flanking
region, exon I, and a short region of intron A was then subcloned into
the HindIII/BamHI sites of Bluescript II
KS
(Stratagene). The sequence of this fragment was
determined on both strands using vector- and insert-specific
oligonucleotide primers. A plasmid containing the 5`-flanking region of
the PI-6 gene (-646 to +31) fused to the chloramphenicol
acetyltransferase (CAT) gene was constructed by digestion of pUC118/7H3
with HindIII (filled) and BspE1 (filled) and ligation
to Bluescript II KS
digested with SmaI. The
resulting plasmid was digested with ClaI and SpeI,
and the insert ligated to a ClaI/XbaI fragment
consisting of the vector and cat gene portion of
pSVTf-CAT(17) .
using Dulbecco's modified Eagle's medium
supplemented with 10% (v/v) fetal bovine serum and 20 mM glutamine (Life Technologies, Inc.). Cells were transfected with
plasmid DNA using the calcium phosphate procedure(12) . Clones
were obtained by co-transfection with a marker plasmid and selection in
G418 as described(17) . Preparation of cell extracts, CAT
assays, the positive control plasmid pSVTf-CAT, and the negative
control plasmid p2CAT has been described previously(17) .
Localization of the Mouse PI-6 Gene
The position
of the mouse PI-6 gene was mapped by analysis of The Jackson Laboratory
(C57BL/B6JEi SPRET/Ei)F1
SPRET/Ei backcross DNA panel
(BSS) as developed and described by Rowe et al.(18) . A PvuII restriction fragment length polymorphism between Mus
musculus (C57BL/B6JEi) and Mus spretus (SPRET/Ei) was
identified by Southern analysis using an XbaI-HindIII
genomic fragment probe containing part of intron A, exon II, and most
of intron B. Approximately 2 µg of genomic DNA from each of the 94
animals in the BSS panel was then digested with PvuII,
separated by agarose gel electrophoresis, transferred to a charged
nylon membrane (GeneScreen Plus, DuPont NEN), and hybridized to the
same probe in 6
SSC at 68 °C. The final wash was in 1
SSC at 68 °C. The presence or absence of the M.
musculus fragment in each of the animals was scored, and the
results were analyzed by L. Rowe at The Jackson Laboratory using the
MapManager program(19) .
Isolation of a cDNA for Mouse PI-6
We have
previously characterized a cDNA encoding human PI-6(7) . This
cDNA was used as a probe in low stringency screens of two mouse liver
cDNA libraries. Although several positive and related clones were
isolated from these libraries, restriction mapping and DNA sequencing
showed that all were incomplete. Comparison of the sequence of the
longest clone to human PI-6 showed 76% similarity at both the
nucleotide and protein level. In particular it showed that the reactive
center residues (P-P`
; Arg-Cys) are conserved
in the mouse molecule, suggesting that it inhibits the same serine
proteinases (e.g. thrombin) as human PI-6. However, in
comparison with the human clone, this mouse clone lacked a
5`-untranslated region and the first 11 codons.
Figure 1:
Sequence of the mouse PI-6 (Spi3) gene. Shown is the composite sequence of the
5`-flanking region and structural gene deduced by analysis of mouse
cDNAs and two genomic clones. Intervening sequences are boxed and shown in lowercase; intron lengths were estimated by
PCR. The transcriptional start site (+1) was deduced by anchored
PCR as described. The TATA box (-24) and polyadenylation signals
(+1292) are underlined. The vertical arrow indicates the putative reactive center bond
(P-P`
), and the asterisk indicates a
potential site for N-linked
glycosylation.
Expression of the Mouse PI-6 cDNA
To show that it
could direct the synthesis of a protein, the mouse PI-6 cDNA was
subcloned into the mammalian expression vector pSVTf(15) . This
vector allows the expression of cDNAs either in mammalian cells under
the control of the SV40 early promoter or in rabbit reticulocyte
lysates following in vitro transcription using T7 RNA
polymerase. In this case, PI-6 mRNA produced in vitro was used
to program a rabbit reticulocyte lysate containing
[S]methionine. Products from the translation
were analyzed by SDS-polyacrylamide gel electrophoresis and
fluorography (Fig. 2). As predicted from the sequence data, a
42-kDa protein was produced in lysates containing mouse PI-6 mRNA (lanes 3 and 4), but was not evident in unprogrammed
lysates (lanes 1 and 2).
Figure 2:
Expression of the mouse PI-6 cDNA. A
full-length cDNA was cloned into the expression vector pSVTf (15) and
linearized at the 3` end of the insert. RNA synthesized in vitro using T7 RNA polymerase was used to program rabbit reticulocyte
lysates containing [S]methionine. 10 ng of
thrombin were added to the indicated samples and incubated at 37 °C
for 30 min. Products were separated by reducing 10% SDS-PAGE and
visualized by fluorography.
To show that the protein
could function as a proteinase inhibitor, thrombin was added to the
lysates prior to SDS-polyacrylamide gel electrophoresis (Fig. 2, lanes 2 and 4). This resulted in the appearance of a
67-kDa species in the programmed lysates. This corresponds to an
SDS-stable complex formed between thrombin and the 42-kDa protein and
is reminiscent of those commonly observed between serpins and their
target proteinases(1) . It also resembles the complex seen
between human PI-6 and thrombin in the reticulocyte system (7) and confirms that mouse PI-6 has inhibitory properties
similar to its human counterpart.
Isolation and Characterization of the Mouse PI-6
Gene
Seven overlapping clones containing fragments of the mouse
PI-6 gene were isolated from a 129/Sv genomic DNA library screened at
high stringency with the full-length mouse cDNA. These clones were
analyzed by EcoRI and HindIII digestion and
hybridization to a panel of mouse PI-6-specific oligonucleotides (PB89,
-90, -91, -96, -97, -98, -99, -104, and -105; ). The entire
gene spanning about 20 kb was represented in two overlapping clones (Fig. 3). Intron/exon boundaries were deduced by direct (cycle)
sequencing of the clones using the oligonucleotide primers shown in and subsequent comparison of the genomic sequences to the
cDNA sequence. This analysis showed that the gene consists of 7 exons
and 6 introns ( Fig. 1and Fig. 3). Clone 5 contains
the exons III-VII and the 3`-flanking region, while clone
7
contains the 5`-flanking region and exons I-V.
Figure 3:
Map of the mouse PI-6 gene. The structure
of the mouse PI-6 gene was deduced by restriction endonuclease mapping,
hybridization with specific oligonucleotide probes (Table I), and DNA
sequencing of two overlapping genomic clones (5 and
7). Exons
are designated by Roman numerals and indicated by filled
boxes. Introns are designated by uppercase letters, and
the TATA box is shown as a closed circle. The RFLP probe is
the segment of genomic DNA used for the gene localization studies. E, EcoRI; H, HindIII.
The length of each
intron was estimated by PCR on genomic clone templates using primers
corresponding to sequences in the flanking exons. The sizes of the
resulting products were measured by comparison to DNA standards in
agarose gel electrophoresis (data not shown). Analysis of the position
and phasing of each intron/exon boundary showed that the PI-6 gene is
identical in every respect with the human PAI-2 and chicken ovalbumin
genes (11) with one exception: the PI-6 gene lacks an
intervening sequence corresponding to the third intron present in both
these genes (see Fig. 8).
Figure 8:
Intron/exon structure of members of the
serpin gene family. This figure is derived from that shown in Ref. 11. Thick lines indicate coding sequences; thin lines indicate untranslated regions; arrows marked by uppercase
letters indicate positions of introns. Sequences are aligned on
the basis of amino acid similarity with the scale showing numbers of
amino acids. AT-III, antithrombin III; 1-AT,
-antitrypsin; PAI-1, plasminogen activator
inhibitor 1; OVAL., ovalbumin; PAI-2, plasminogen
activator inhibitor 2; SPI3/PI6, proteinase inhibitor
6.
Localization and Designation of the Mouse PI-6
Gene
Recently, Rowe et al.(18) have described a
panel of DNAs derived from interspecific backcross mice that can be
used for mapping genetic loci in the mouse. Maps have been generated
using these DNAs and anchored to other published maps. To place an
unknown gene on the map, a specific PCR or restriction fragment length
polymorphism between the parental species of mice (M. musculus C57BL/6JEi and M. spretus SPRET/Ei) must be found. The
distribution of the polymorphism in DNA from 94 (M. musculus M. spretus)F1
M. spretus (BSS)
or 94 (M. musculus
M. spretus)F1
M. musculus (BSB) backcrossed mice is then analyzed to deduce
the map position.
gene (39 centimorgans distal).
Figure 4:
Chromosome linkage map showing the
location of the mouse PI-6 gene. The midregion of mouse chromosome 13
is shown as a solid line. Markers in this region of the
chromosome are indicated; those in the flanking regions (dashed
lines) are not shown. Those prefixed with D13 are motif-primed PCR
markers (18). Pl1, placental lactogen gene; Ctla2a, ctla2 gene; Tpbp, trophoblast-specific protein
gene.
The
International Committee on Standardized Genetic Nomenclature for Mice
has recommended that the mouse homologue of human PI-6 be designated
proteinase inhibitor 3, with a gene Spi3.(
)
Analysis of the Promoter Region of the Mouse
PI-6 Gene
In one of the genomic clones (7), we identified
sequences corresponding to the 5` end (exon I) of the mouse cDNA. This
clone also contains approximately 650 bp of flanking sequences which
are likely to comprise the promoter region of the PI-6 gene. DNA
sequence analysis of this region (see Fig. 1) identified a
potential TATA box at nucleotide -24 (TATTTAA).
Figure 5:
The
5`-flanking region contains a functional promoter. A 680-bp fragment
(-650 to +31) of the mouse PI-6 gene was placed in front of
the CAT gene in the plasmid pPI-CAT. Clones of BALB/c 3T3 cells
carrying this plasmid (or the control plasmids pSVtf-CAT and p2CAT)
were derived by co-transfection with a marker plasmid and selection in
G418. Extracts from comparable numbers of pooled clones were normalized
on protein and assayed for CAT activity by thin layer chromatography
analysis of [C]chloramphenicol acetylation. Arrow indicates direction of migration of solvent front; or. indicates origin.
Expression of the Mouse PI-6 Gene in Adults and
Embryos
To analyze the expression of the mouse PI-6 gene, we
developed a reverse transcriptase-PCR strategy. Tissues and organs were
dissected from adult or 15-day-old embryonic mice. Total RNA was
prepared and used as template in a reverse transcription reaction
primed by oligo(dT). The resulting cDNA was then used for PCR. Each PCR
included two sets of primers: one pair to amplify PI-6 sequences and
the other pair to amplify glyceraldehyde-3-phosphate dehydrogenase
sequences (). The glyceraldehyde-3-phosphate dehydrogenase
primers were included as normalization controls and to check the
integrity of the RNA/cDNA in tissues lacking PI-6. The PCR products
were analyzed on ethidium bromide-stained agarose gels and then
transferred to nylon membranes for Southern hybridization using a
PI-6-specific oligonucleotide probe.
Figure 6:
Analysis of PI-6 mRNA levels in the adult
mouse. Total RNA isolated from the tissues of an adult mouse was used
as template for cDNA synthesis with (+) or without (-)
reverse transcriptase (RT). A portion of the cDNA reaction was
subsequently added to a PCR containing mouse PI-6-specific primers and
mouse glyceraldehyde-3-phosphate dehydrogenase-specific primers.
Products were separated on a 2% ethidium bromide agarose gel (upper
panel), then transferred to a nylon membrane, hybridized to
oligonucleotide PB-92, and visualized by autoradiography (lower
panel). Lanes 1, heart; 2, brain; 3,
spleen; 4, lung; 5, liver; 6, muscle; 7, kidney; and 8, testis.
Figure 7:
Analysis of PI-6 mRNA levels in 15-day
mouse embryos. Total RNA isolated from the pooled tissues of five
15-day embryos was used as template for cDNA synthesis with (+) or
without (-) reverse transcriptase (RT). A portion of the
cDNA reaction was subsequently added to a PCR containing mouse
PI-6-specific primers and mouse glyceraldehyde-3-phosphate
dehydrogenase-specific primers. Products were separated on a 2%
ethidium bromide agarose gel (upper panel), then transferred
to a nylon membrane, hybridized to oligonucleotide PB-92, and
visualized by autoradiography (lower panel). Lanes 1,
yolk sac; 2, placenta; 3, liver; 4, heart; 5, gut; 6, kidney; 7, lung; 8,
thymus; 9, brain; 10, muscle; and 11, ES
cells.
(
)Sequence comparison shows
that there is no conservation of the number or position of potential
sites for asparagine-linked glycosylation in the human (3 sites) or
mouse (1 site) molecules. We consider it unlikely that these molecules
are secreted and propose that they fulfill an intracellular role. An
intracellular function for serpins is not unprecedented as it is clear
that the cytosolic viral serpin CrmA prevents apoptosis by inhibiting
an interleukin 1
converting enzyme-like cysteine
proteinase(20, 21) .
-1 proteinase inhibitor, and
-antichymotrypsin have similar gene organizations and
are clustered on chromosome 14(10) . In the mouse there are two
loci on chromosome 12 that contain multiple serpin genes; the Spi1 locus consisting of at least 4 serpin genes and the Spi2 locus of 12 or more genes resembling
-antichymotrypsin(24, 25) .
Table: Oligonucleotides used as probes, sequencing
primers, and PCR primers
Table: Mapping of the
mouse Spi3 (PI-6) gene to chromosome 13 in a (M. musculus M.
spretus) F1
M. spretus backcross typing panel
/EMBL Data Bank with accession number(s) U25843 and U25844.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.