(Received for publication, December 8, 1995; and in revised form, January 12, 1996)
From the
There is growing evidence that mammalian AMP-activated protein
kinase (AMPK) plays a role in protecting cells from stresses that cause
ATP depletion by switching off ATP-consuming biosynthetic pathways. The
active form of AMPK from rat liver exists as a heterotrimeric complex
and we have previously shown that the catalytic subunit is structurally
and functionally related to the SNF1 protein kinase from Saccharomyces cerevisiae. Here we describe the isolation and
characterization of the two other polypeptides, termed AMPK and
AMPK
, that together with the catalytic subunit (AMPK
) form
the active kinase complex in mammalian liver. Sequence analysis of cDNA
clones encoding these subunits reveals that they are related to yeast
proteins that interact with SNF1, providing further evidence that the
regulation and function of AMPK and SNF1 have been conserved throughout
evolution. The amino acid sequence of the
subunit is most closely
related to SIP2 (35% identity), while the amino acid sequence of the
subunit is 35% identical with SNF4. We show that both AMPK
and AMPK
mRNA and protein are expressed widely in rat tissues. We
show that AMPK
interacts with both AMPK
and AMPK
in
vitro, whereas AMPK
does not interact with AMPK
under
the same conditions. These results suggest that AMPK
mediates the
association of the heterotrimeric AMPK complex in vitro, and
will facilitate future studies aimed at investigating the regulation of
AMPK in vivo.
A number of recent studies have led to the proposal that in
mammals an AMP-activated protein kinase (AMPK) ()plays a
major role in the response to metabolic stress (Corton et al.,
1994; Hardie, 1994; Hardie et al., 1994). AMPK was first
identified through its role in the phosphorylation and inactivation of
a number of enzymes involved in lipid metabolism (Carling et
al., 1987; Hardie et al., 1989; Hardie, 1992), and
subsequently was shown to phosphorylate enzymes in other metabolic
pathways (Carling and Hardie, 1989). AMPK has been purified from a
number of species, including human, rat, and pig, and in each case is
activated allosterically by micromolar concentrations of AMP (Carling et al., 1989; Mitchelhill et al., 1994; Sullivan et al., 1994). The kinase is itself regulated by reversible
phosphorylation, being phosphorylated and activated by a distinct AMPK
kinase (AMPKK), thereby forming a protein kinase cascade (Carling et al., 1987; Weekes et al., 1994). The
phosphorylation and activation of AMPK is markedly stimulated by AMP
(Moore et al., 1991; Hawley et al., 1995), making
AMPK extremely sensitive to changes in the intracellular concentration
of AMP. These findings have led to the proposal that one of the primary
roles of AMPK is to conserve ATP during periods of excessive ATP
utilization, when AMP levels are elevated (Corton et al.,
1994; Hardie et al., 1994).
We recently reported that the deduced amino acid sequence of the catalytic subunit of rat liver AMPK is remarkably similar to the sequence of the yeast protein kinase SNF1 (Carling et al., 1994). In a further study we went on to show that SNF1 is functionally related to mammalian AMPK (Woods et al., 1994). In vitro, SNF1 phosphorylates a specific peptide substrate for AMPK, and there is good evidence that SNF1 phosphorylates and inactivates acetyl-CoA carboxylase in vivo. Furthermore, like AMPK, SNF1 is inactivated by protein phosphatases and can be reactivated by a partially purified preparation of mammalian AMPKK, suggesting functional conservation of the upstream kinases (Woods et al., 1994). The SNF1 protein kinase from Saccharomyces cerevisiae is required for the expression of glucose repressed genes in response to glucose starvation (Celenza and Carlson, 1986; Estruch et al., 1992; Gancedo, 1992), e.g. the SUC2 gene, which encodes invertase (Carlson and Botstein, 1982). snf1 mutants are unable to utilize a wide range of non-glucose sugars (Carlson et al., 1981; Estruch et al., 1992). In addition, snf1 mutants have been shown to be defective in other aspects of cell growth, e.g. glycogen synthesis and sensitivity to heat stress (Thompson-Jaeger et al., 1991). SNF1 is physically associated with a 36-kDa polypeptide, termed SNF4 (Celenza et al., 1989), which is itself required for expression of many glucose-repressible genes. SNF4 is thought to function as an activator of SNF1 (Celenza and Carlson, 1989; Celenza et al., 1989), although the mechanism by which SNF4 activates SNF1 is not known.
A number of yeast proteins, which interact with SNF1 in vivo, termed SNF1 interacting proteins or SIPs, have been identified using the two-hybrid system (Yang et al., 1992). Two of these proteins, SIP1 and SIP2, share significant amino acid sequence identity, particularly at their C termini (Yang et al., 1992). Furthermore, the amino acid sequence of SIP2 is 52% identical to GAL83 (Erickson and Johnston, 1993; Yang et al., 1994). GAL83 is involved in the glucose repression of GAL genes, and genetic evidence suggests that GAL83 is involved in the SNF1 pathway (Matsumoto et al., 1981; Erickson and Johnston, 1993). SIP1, SIP2, and GAL83 have been shown to co-immunoprecipitate with SNF1, and all three proteins are phosphorylated in an immune complex SNF1 kinase assay (Yang et al., 1994). In the same study it was shown that the C-terminal 80 amino acids of SIP2, termed the ASC domain, were sufficient to mediate interaction with SNF1. The functions of SIP1, SIP2, and GAL83 remain unknown, although it has been proposed that they may act as modulators of SNF1, targeting the kinase to specific intracellular locations and/or substrates (Yang et al., 1994).
Recently, AMPK has
been purified to apparent homogeneity from both rat and pig liver
(Mitchelhill et al., 1994; Davies et al., 1994). Two
other polypeptides co-purified with the catalytic subunit (molecular
mass 63 kDa), and biochemical analysis of the purified kinase complex
indicated that AMPK isolated from rat liver exists as a heterotrimer
(Davies et al., 1994). We subsequently reported that the
catalytic subunit of AMPK isolated from rat skeletal muscle did not
appear to be associated with any other polypeptides and that this
observation might account for the low activity of AMPK detectable in
skeletal muscle (Verhoeven et al., 1995). In this paper we
report the isolation and cDNA cloning of two AMPK subunits from rat
liver which we refer to as AMPK and AMPK
(the catalytic
subunit is designated AMPK
) following the terminology of Kemp and
colleagues (Stapleton et al., 1994). The
subunit is most
closely related to SIP2 and contains a region at its C terminus, which
is 50% identical with the ASC domain of SIP1/SIP2/GAL83 (Yang et
al., 1994). The
subunit has a high degree of amino acid
sequence identity with SNF4, and this conservation of sequence suggests
that, like SNF4, it is necessary for the catalytic activity of AMPK. We
show here that AMPK
interacts with both AMPK
and AMPK
and that this mediates the assembly of the ternary complex in
vitro. The similarity between the mammalian AMPK complex and the
SNF1 complex from yeast emphasizes the likelihood that the role of
these kinases have been highly conserved throughout evolution.
Autophosphorylated AMPK was prepared by incubating the immune
complex with 0.2 mM [-
P]ATP and
0.2 mM AMP at 30 °C for 30 min. Unincorporated ATP was
removed by extensive washing with buffer A. Protein was eluted from the
resin as above. Following SDS-PAGE the gel was dried and subjected to
autoradiography at -70 °C.
In order to obtain the 5` end of the cDNA
encoding the subunit, antisense oligonucleotide primers were
synthesized based on
cDNA sequence and used to perform 5`
RACE-PCR (Frohman et al., 1988). (AMPK
: RACE primer 1,
TGGCGTAGGTGCCAATCTG; RACE primer 2, ATCTGTAGCTCTTCCAGAG). Rat liver
RACE-ready cDNA (Clontech) was used as a template for amplification
using primer 1 and the anchor primer for 30 cycles of 94 °C, 1 min;
58 °C, 1 min; 72 °C, 1 min. A second round of amplification on
an aliquot (0.1 µl) of the reaction products was performed using
primer 2 and the anchor primer under the same conditions as before.
Products from the second round of amplification were isolated by
agarose gel electrophoresis, cloned into pGEM-T vector, and sequenced.
In order to construct a cDNA encoding the entire amino acid sequence of
the
subunit oligonucleotide primers spanning the initiating
methionine (GCCAAGGTCGACGGCCGGGTGCTAGCAATG) and downstream of
the stop codon (GGCCACTAGTCGACTCCGTTCTCTCAGG) and containing a SalI restriction site (underlined) were synthesized and used
to amplify rat liver cDNA. The product (1.1 kb) was cloned into pGEM-T
vector to yield pGEM-
. The inserts from each of three independent
clones were sequenced to confirm their authenticity.
Yeast (strain
SFY526 harboring a GAL1-lacZ reporter gene) were transformed
with various combinations of the vectors and grown on selective media.
To test for interactions, several colonies from each transformation
were patched onto selective plates and grown for 2 days at 30 °C.
Colonies were transferred to nitrocellulose filters and cells were
permeabilized by freeze-thawing in liquid nitrogen. The filters were
incubated in the presence of X-Gal at 30 °C for 1-2 h in
order to determine any blue coloration. For quantitative analysis,
transformants were grown to mid-log phase in selective liquid culture
and -galactosidase activity was determined in permeabilized cells.
In every case, activities were measured in at least two transformants
and assays were performed in triplicate. Values are expressed in Miller
units (Miller, 1972) with a standard error of less than 20% of the
mean.
Figure 1:
Co-purification of two
polypeptides with the catalytic subunit of rat liver AMPK. Partially
purified AMPK from rat liver was purified using protein A-Sepharose
cross-linked to affinity-purified antibodies raised against AMPK.
Following extensive washing, the antibody-protein A-Sepharose resin was
incubated with 0.2 mM [
-
P]ATP and
0.2 mM AMP at 30 °C for 30 min. Proteins were eluted from
the antibody-protein A-Sepharose resin, resolved by SDS-PAGE, and
visualized by staining with Coomassie Blue (lane A). An
autoradiograph of the same gel is shown in lane B. The
migration of molecular mass standards is shown on the right of
the figure.
Figure 2:
Nucleotide sequence and predicted amino
acid sequence of AMPK and AMPK
. A, AMPK
. The
initiating codon and stop codon are shown in bold, and peptide
sequences derived from purified AMPK
are underlined. An
in-frame stop codon upstream of the initiating methionine is shown in bold, and a potential polyadenylation signal sequence is underlined. Nucleotides are numbered on the right and amino acids on the left. B,
AMPK
.
Figure 3:
In vitro translation of
AMPK. [
S]Methionine-labeled AMPK
was
translated in vitro in rabbit reticulocyte lysate programmed
with RNA synthesized from AMPK
cDNA cloned into pET-14b. The
lysate was immunoprecipitated using anti-
antibodies attached to
protein A-Sepharose and the immune complex analyzed by SDS-PAGE. AMPK
was immunoprecipitated from rat liver with anti-
antibodies and
resolved by SDS-PAGE on the same gel in order to compare the
electrophoretic mobility of the native and recombinant
subunits.
Proteins were visualized by staining with Coomassie Blue, and labeled
products were detected by fluorography. Lane 1, total lysate
in the absence of RNA; lane 2, total lysate programmed with
AMPK
RNA; lane 3, immunoprecipitation of AMPK from rat
liver; lane 4, immunoprecipitation of lysate programmed with
AMPK
RNA. Note that in lane 3 all three AMPK subunits are
precipitated and that AMPK
is the upper polypeptide in the
38/36-kDa doublet. The migration of molecular mass markers is shown on
the left.
Figure 4:
AMPK and AMPK
are related to
yeast proteins that interact with SNF1. A, the deduced amino
acid sequences of AMPK
(top) and SIP2 (bottom)
were aligned using the GAP program in the University of Wisconsin
package with a gap weight of 3.0 and a length weight of 0.1. Dots indicate gaps introduced to maximize the alignment. Identities
between the two sequences are shown in shaded boxes. B, the deduced amino acid sequences of AMPK
(top) and SNF4 (bottom) were aligned as
above.
Figure 5:
Northern blot analysis of AMPK,
, and
mRNA. Approximately 2 µg of poly(A)-rich RNAs
isolated from the indicated tissues were separated on a 1.2% agarose
gel under denaturing conditions, transferred to a charged-modified
nylon membrane, and probed separately with either a 1.9-kb fragment of
AMPK
cDNA or a 1.1-kb fragment of AMPK
cDNA. In each case the
blot was washed under stringent conditions (0.2
SSC, 0.5% SDS
at 65 °C) and exposed for either 2 days (AMPK
) or 5 days
(AMPK
) at -70 °C. For comparison a Northern blot of the
subunit is shown (Verhoeven et al., 1995). The migration
of RNA markers are indicated.
Antibodies raised against fusion proteins of AMPK or AMPK
with glutathione S-transferase were used to determine the
expression of the polypeptides in various rat tissues. We also examined
the expression of AMPK
(the catalytic subunit) using antibodies
raised against
specific peptides (Carling et al., 1994). Fig. 6shows the expression of the polypeptides in a number of
tissue lysates. All three polypeptides were detected in every tissue
tested, although there appeared to be some variation in the relative
amounts of the three subunits present in different tissues (for
instance compare the expression of the
and
subunits in
brain and skeletal muscle). As we have noted previously, there is a
small but detectable shift in the mobility of the
subunit between
different tissues, which we believe may reflect differences in the
phosphorylation state of the enzyme (Verhoeven et al., 1995).
Figure 6:
Western blot analysis of AMPK,
,
and
. Approximately 100 µg of tissue lysate (14,000
g supernatant) from the indicated tissues were separated by
SDS-PAGE and transferred to a polyvinylidene membrane. Separate blots
were probed with polyclonal antibody to AMPK
, AMPK
, or
AMPK
. Primary antibody was detected using a goat anti-rabbit
antibody conjugated to horseradish peroxidase and visualized by
enhanced chemiluminescence.
Since the two-hybrid system is carried out
in yeast, it was important to determine any interactions between the
mammalian subunits and their yeast counterparts. We therefore extended
the study to determine interactions between AMPK subunits and SNF1,
SNF4, SIP1, and SIP2. Table 3shows the results of the various
combinations of rat and yeast proteins in the two-hybrid system. We
were unable to detect any interaction of AMPK with any of the
yeast proteins. However, AMPK
gave a signal with both SNF1 and
SNF4 and AMPK
interacted with SNF1, SIP1, and SIP2. Furthermore,
AMPK
interacted with all of the SIP1 and SIP2 fusions tested,
including a fusion expressing the C-terminal 120 amino acids of SIP2
(G
-SIP2
; Yang et al. (1994)).
Figure 7:
Association of AMPK subunits in
vitro. A, [S]methionine-labeled
AMPK subunits were generated in rabbit reticulocyte lysates programmed
with the indicated RNAs and an aliquot of the products was analyzed by
SDS-PAGE and fluorography. The AMPK
RNA used for these studies
lacked sequence encoding the N terminus. AMPK
, therefore, migrates
with a lower apparent molecular mass compared to AMPK
, allowing
the subunits to be clearly resolved. The migration of molecular mass
markers is shown on the left. B, interaction of the
subunits was determined by co-immunoprecipitation from the lysates (2
volume used in A). Following incubation with preimmune
serum and protein A-Sepharose, lysates were immunoprecipitated with
subunit-specific antibodies as indicated. The immune complexes were
washed extensively (as described under ``Materials and
Methods''), boiled with SDS-sample buffer, and resolved by
SDS-PAGE. Labeled products were detected by
fluorography.
The results of the in vitro translation
studies demonstrate that the and
subunits and the
and
subunits interact with each other forming relatively stable
complexes, whereas there is no evidence for a stable complex between
the
and
subunits. If the
and
subunits do
interact, then their association must be either transient or weak, or
both, and does not survive immunoprecipitation. The translations and
immunoprecipitations are carried out in buffers lacking protein
phosphatase inhibitors and under conditions that would not be expected
to cause activation of endogenous AMPKK, which may be present in the
reticulocyte lysate. It is likely that AMPK
is in the
dephosphorylated form following translation, suggesting that
phosphorylation by AMPKK is not necessary for formation of the ternary
complex. This may also explain why we have been unable to detect AMPK
activity in any of the translations.
Full-length cDNA clones encoding AMPK were isolated by
conventional library screening, and a composite cDNA clone encoding the
full-length sequence of AMPK
was constructed from overlapping
clones isolated by a combination of library screening and 5` RACE. The
deduced amino acid sequence of AMPK
predicts a protein with a
molecular mass of 30 kDa. This is considerably lower than the apparent
mass of the
subunit isolated from rat liver, as judged by
SDS-PAGE. In vitro translation of RNA synthesized from
AMPK
cDNA produced a major product, which exactly co-migrated with
rat liver AMPK
following SDS-PAGE. This finding, coupled with the
fact that an in-frame stop codon is present upstream of the first
methionine, confirms that the AMPK
cDNA reported here is
full-length. The reason for the anomalous electrophoretic mobility of
the
subunit in denaturing gels is unclear, but if it is due to
post-translational modification of the polypeptide the reticulocyte
lysate system must be competent in carrying out the modification.
The finding that AMPK and AMPK
share sequence identity
with yeast proteins that interact with SNF1 strengthens the proposal
that the functions of the two kinases have been highly conserved
throughout evolution (Woods et al., 1994). Although the
function of SNF4 is not known, it is necessary for the protein kinase
activity of SNF1 and it seems likely that AMPK
will have a similar
role in the activity of AMPK. Despite the obvious similarity between
the amino acid sequences of AMPK
and SNF4, we have not been able
to complement snf4 mutants by expression of AMPK
. (
)In a previous study, we reported that we were unable to
complement snf1 mutants by expression of the catalytic subunit
of AMPK, which shares 47% amino acid sequence identity with SNF1
(Carling et al., 1994; Woods et al., 1994). Taken
together these results indicate that, although the AMPK and SNF1
complexes are highly related, significant differences between the two
complexes must exist. One notable difference that is already known is
that, while the mammalian kinase is markedly activated by AMP (Carling et al., 1989), no measurable effect of AMP on SNF1 activity
has been demonstrated (Mitchelhill et al., 1994; Woods et
al., 1994). Whether this difference alone is sufficient to explain
the inability of the catalytic subunit to rescue snf1 mutants,
and AMPK
to rescue snf4 mutants, is not yet clear. In
order to address this question, a detailed comparison of the structures
of the mammalian and yeast kinase complexes, e.g. from
crystallographic studies, and the elucidation of the regulation of the
kinases are required.
Western blot analysis of rat tissue lysates
shows that AMPK and AMPK
subunits are expressed in every
tissue examined. Although the blots are not quantitative, there does
appear to be some variation in the relative expression of the three
subunits in different tissues (Fig. 6). AMPK purified from rat
liver appears to exist entirely as a heterotrimeric complex of
AMPK
,
, and
(see Fig. 1and Davies et
al.(1994)). Immunoprecipitation of
from skeletal muscle,
however, suggests that it exists predominantly as a monomer, with no
kinase activity (Verhoeven et al., 1995), even though
AMPK
and AMPK
are present in this tissue. These findings
raise the interesting possibility that the association of the catalytic
subunit with AMPK
and AMPK
is regulated and that the
structure of the complex may vary between different tissues and/or
different conditions. At present the mechanism that leads to the
association of the subunits is not known, although our results suggest
phosphorylation is not required. Dephosphorylation of the active AMPK
complex to an inactive form in vitro does not result in
dissociation of the subunits (data not shown), and it is interesting to
note that in yeast the association of SIP1 or SIP2 with SNF1 does not
require SNF1 protein kinase activity or the presence of SNF4 (Yang et al., 1994).
The results from the two-hybrid experiments
indicate that AMPK interacts with both AMPK
and AMPK
,
but that the interaction of AMPK
with AMPK
is very weak. We
also tested the interaction of the mammalian subunits with their yeast
counterparts in the two-hybrid system. In this case we found evidence
for the interaction of AMPK
with both SNF1 and SNF4 and AMPK
with SNF1 and SIP1 and SIP2. However, there was no detectable
interaction between AMPK
and any of the yeast proteins. These
results do not rule out the possibility that the AMPK
-SNF1
interaction is indirect and could be mediated by an AMPK
homologue
in yeast, e.g. SIP1/SIP2/GAL83, which could act as a bridging
protein in a ternary complex.
The results from the in vitro study show that AMPK interacts with both AMPK
and
AMPK
forming stable complexes. However, under the same conditions,
we could not detect stable complexes between AMPK
and AMPK
.
These results suggest that the formation of the ternary complex between
,
, and
is mediated by the
subunit. We have not
been able to detect AMPK activity in lysates programmed with all three
subunits, or in immune complexes from these lysates. This may be due to
the dephosphorylated form of the kinase and/or the lack of sufficient
protein in the translation system to allow detection of kinase
activity.
Two different models for the association of the three
subunits can be predicted based on the results of this study. The first
model is one in which the subunit links the
and
subunits (Fig. 8A). The second model involves a
conformational change in either the
or
subunit, or both,
upon binding of the
subunit, which would then allow direct
interaction between the
and
subunits (Fig. 8B). It is interesting to note the similarities
between the AMPK complex and the heterotrimeric complex formed between
CDK-activating kinase, cyclin H, and p36/MAT1 (RING finger protein)
(Fisher et al., 1995; Devault et al., 1995). Although
there is no significant amino acid sequence identity between the AMPK
subunits and the CDK-activating kinase subunits, the regulation and
association of the two kinase complexes bear some obvious resemblances.
Figure 8:
Model for the association of an active
AMPK complex. Our results demonstrate that AMPK interacts with
both AMPK
and AMPK
and that this mediates the formation of a
stable trimeric complex. AMPK
could act as a bridge linking the
other two subunits (A), or alternatively AMPK
could cause
a conformational change in AMPK
, AMPK
, or both (depicted by
the different labeling of the subunits) allowing the
and
subunits to interact (B). AMPK
is phosphorylated by an
upstream kinase (AMPKK), leading to activation of the complex (Weekes et al., 1994; Hawley et al., 1995). Our preliminary
results indicate that AMPKK can only phosphorylate AMPK
when it is
in the heterotrimeric form, implying that association of the complex is
a prerequisite for phosphorylation. AMP activates the complex both
allosterically and by promoting the phosphorylation of AMPK
by
AMPKK, although it is not known whether AMP has any direct effect on
the association of the complex.
Although the functions of AMPK and AMPK
remain unclear, a
possible insight can be gained by comparison with a proposed model for
the SNF1 complex in yeast. SNF4 is necessary for SNF1 kinase activity in vitro (Woods et al., 1994) and may therefore
fulfill a similar function to cyclins in the activation of cyclin
dependent kinases (Jeffrey et al., 1995). AMPK
by analogy
would play a similar role in the activation of AMPK. Biochemical
evidence suggests that SNF1 forms a relatively stable complex with
SNF4, and that this complex has protein kinase activity in vitro (Mitchelhill et al., 1994). The SNF1
SNF4 complex
interacts with one of a number of related proteins, which include SIP1,
SIP2, and GAL83 (Yang et al., 1994). It has been proposed that
these proteins act as adaptors or targeting subunits, directing the
kinase to specific intracellular substrates (Yang et al.,
1994). In this model, the formation of different
SNF1
SNF4
adaptor complexes would allow selective
phosphorylation of the downstream targets of SNF1, if the adaptor
proteins recognize different substrates. It is not clear whether the
assembly of the SNF1
SNF4
adaptor complex is regulated, or
how the adaptor proteins act to promote phosphorylation of target
substrates. However, there is clear evidence from other systems that
one mechanism for regulating the phosphorylation of a protein is to
regulate the distribution of the kinase, or phosphatase, acting on that
protein via specific targeting subunits (Hubbard and Cohen, 1993;
Coghlan et al., 1995). Could AMPK
act as a targeting
subunit for the AMPK complex? It seems unlikely that the function of
AMPK
is merely to bring together the
and
subunits,
especially given the fact that in yeast there appears to be a family of
proteins related to AMPK
. It will be interesting to determine
whether or not there is a family of proteins related to AMPK
in
mammalian cells, and whether these proteins act as adaptors for AMPK.
Finally, it is interesting to note that, although the interaction of
SNF1 and SNF4 is often used as a model for the two-hybrid system, our
results imply that this interaction could in fact be indirect. Given
the similarities between the mammalian AMPK and yeast SNF1 complexes it
is possible that the interaction between SNF1 and SNF4 is mediated by a
member of the SIP1/SIP2/GAL83 family of proteins. In addition to SNF4,
a number of other polypeptides were found to co-purify with SNF1
(Stapleton et al., 1994). Although SIP1 was not identified, it
is possible that some of these co-purifying polypeptides are members of
the SIP1/SIP2/GAL83 family, which could mediate the association of SNF1
and SNF4 in a ternary complex, analogous to AMPK in the mammalian
complex.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X95577 [GenBank]and X95578[GenBank].