(Received for publication, October 27, 1994; and in revised form, January 9, 1995)
From the
We report our studies on the characterization of an 14-kDa
lectin, termed galectin-1, that we have found to be expressed by
Chinese hamster ovary (CHO) cells. cDNA for galectin-1 from CHO cells
was prepared and sequenced, and a recombinant form (rGal-1) was
expressed in Escherichia coli. A mutated form of the protein
that fully retained activity was also constructed (termed C2SrGal-1) in
which Cys-2 was changed to Ser-2. rGal-1 was stable in the presence of
reducing agent, but it quickly lost all activity in the absence of
reducing agent. In contrast, glycoprotein ligands, such as basement
membrane laminin, stabilized the activity of rGal-1 in the absence of
reducing agent (t
= 2 weeks). C2SrGal-1
was stable in the presence or absence of either ligand or reducing
agent. Unexpectedly, galectin-1 was found to exist in a reversible and
active monomer-dimer equilibrium with a K
7 µM and an equilibration time of t
10 h. Addition of haptenic sugars did not
affect this equilibrium. Galectin-1 isolated from the cytosol of CHO
cells was found to exist as monomers and dimers. These studies
demonstrate that galectin-1 binding to a biological ligand stabilizes
its activity and that the monomer/dimer state of the protein is
regulated by lectin concentration.
A variety of vertebrate cells and tissues synthesize a class of
-galactoside binding proteins termed galectins, which occur in low
(galectin-1 and -2) and high molecular weight forms (galectin-3 and -4)
(Barondes, 1984; Barondes et al., 1994). Recently, galectins
have also been found in lower invertebrates, such as sponge and
nematode (Hirabayashi et al., 1992a, 1992b; Pfeifer et
al., 1993). Galectins form a family of proteins (Gitt and
Barondes, 1986) and are distinct from other animal lectins such as the
Ca
-dependent lectin family (C-type lectins)
(Drickamer, 1988).
There have been several peculiar observations
made about galectin-1 from diverse sources. (i) Galectin-1 is found
mainly in the cytosol of most cells in which it occurs (Briles et
al., 1979), (ii) the lectin has free sulfhydryls and is inactive
in the absence of reducing agents (Hirabayashi and Kasai, 1991;
Barondes, 1984), (iii) the lectin lacks an identifiable signal sequence
and does not appear to be secreted through the normal secretory pathway
in differentiated myoblasts (Cooper and Barondes, 1990), (iv) the
lectin binds to -galactosyl-containing glycoconjugates on the cell
surface and in the extracellular matrix (Cerra et al., 1984;
Merkle and Cummings, 1988; Do et al., 1990; Cooper et
al., 1991; Zhou and Cummings, 1993), and (v) galectin-1 is a dimer
but disulfides are not involved (Liao et al., 1994).
These facts about galectin-1 raise many questions about its structure and function. If the lectin requires reducing agents to maintain its activity, how can the lectin function outside the cells and in the extracellular matrix? How is the activity of the extracellular form regulated? Does the lectin occur in some cases as a monomer or even higher oligomers? What structural forms of the lectin occur in the cytosol? Does the extracellular form of the lectin arise through secretion or through cellular injury?
These questions about the
structure and function of galectin-1 have led to the present studies.
We have discovered that Chinese hamster ovary (CHO) ()cells
synthesize relatively large amounts of galectin-1 and have cloned and
sequenced the cDNA for the lectin. A recombinant form of the protein
was prepared to aid in understanding its structure. Contrary to
expectations, we found that the lectin is highly stable in the absence
of reducing agents when associated with a biological ligand, such as
laminin. Furthermore, galectin-1 exists in a reversible monomer-dimer
equilibrium. CHO cells are a convenient cell type in which to study the
structure and function of galectin-1. The wild type CHO cells produce
poly-N-acetyllactosamine-containing glycoconjugates bound by
galectin-1, whereas some mutant CHO cell lines resistant to the
cytotoxic effects of plant lectins lack glycoconjugates bound by
galectin-1 and quantitatively secrete the protein. In the accompanying
manuscript (Cho and Cummings, 1995), we take advantage of this system
to study the biosynthesis and localization of galectin-1 and the role
of glycoconjugates in these processes.
E.
coli strain M15 (Qiagen) were transformed with these plasmids to
express recombinant galectin-1 (rGal-1) and C2S galectin-1 (C2SrGal-1)
at high levels. rGal-1 and C2SrGal-1 were purified from sonicated E. coli cell extracts on a column of asialofetuin-Sepharose
(1.5 30 cm). The column was washed with 5 column volumes of
SPB-azide (6.7 mM KH
PO
, 150 mM NaCl, pH 7.4, containing 14 mM 2-mercaptoethanol and
0.02% NaN
) and 3 volumes of SPB-azide containing 1 M NaCl. The bound rGal-1 or C2SrGal-1 were eluted with SPB-azide
containing 0.1 M lactose and dialyzed against SPB-azide.
Approximately 30 ml of rGal-1 (1.13 mg/ml) and 30 ml of C2SrGal-1 (1.3
mg/ml) were obtained from each 1-liter culture of transfected E.
coli. To stabilize the rGal-1 against oxidative inactivation, all
buffers used during purification contained
-mercaptoethanol.
S-Labeled rGal-1 and C2SrGal-1 were produced by growing E. coli in the minimal media (1 liter) containing
Na
SO
(10 mCi) until the A
reached 0.5. An equal volume of methionine-deficient minimal
essential media (1 liter) containing 1 mCi of
S-Protein
Labeling Mix and isopropyl-1-thio-
-D-galactopyranoside (2
mM) was added, and the cells were incubated for another 5 h
with shaking. The E. coli were harvested and lysed by
sonication, and the extracts were applied to a column of
asialofetuin-Sepharose (1.5
30 cm containing 10 mg/ml
conjugated protein and a total volume of 100 ml) to purify radiolabeled
rGal-1 or C2SrGal-1. Specific activity of purified C2SrGal-1 and rGal-1
were 10,922 cpm/µg and 9,745 cpm/µg, respectively. Protein
concentration was determined by the BCA assay.
Figure 1: The cDNA sequence and encoded amino acid sequence of galectin-1 from CHO cells compared to rat galectin-1.
To allow more definitive biochemical characterization and
generate a more stable form of galectin-1, we also constructed a
mutated form of the protein in which the conserved Cys-2 residue was
converted to Ser-2, as described under ``Experimental
Procedures.'' This form of the lectin was termed C2SrGal-1. It has
been shown that Cys residues are not critical for lectin activity but
they contribute to instability of the protein in the absence of
reducing agents (Hirabayashi and Kasai, 1991; Abbott and Feizi, 1991).
We constructed two expression plasmids using pQAE 50 and cDNA for
galectin-1 (rGal-1) and cDNA for mutated galectin-1 (C2SrGal-1). Both
transformed E. coli strains produced the recombinant lectins
in the presence of 2 mM isopropyl-1-thio--D-galactopyranoside. These
recombinant lectins had specific activities similar to the native
galectin-1, in both hemagglutinating assays and binding assays to
immobilized asialofetuin and laminin.
Figure 2: Demonstration of antibody specificity by Western blotting. Monospecific antibodies (80 µg/ml) toward galectin-1 were obtained from rabbit sera after purification on a column of rGal-1-Affi-Gel 15 (1 mg/ml), as described under ``Experimental Procedures.'' CHO cells extracts (50 µg) were analyzed by SDS-PAGE and transferred to nitrocellulose. Strips from a single blot were cut and stained with a monospecific antibody (1:10 dilution) (A) and corresponding preimmune rabbit IgG (B). (Migration positions are indicated for protein molecular mass standards of 200, 116, 94, 68, 43, 29, and 14 kDa.)
Figure 3:
Stability of the rGal-1 and C2SrGal-1
under various conditions. A, the hemagglutination activity of
rGal-1, stored in the absence or presence of reducing solutions, was
assayed over a period of 48 h, as described under ``Experimental
Procedures.'' The hemagglutinating activity of the lectin is
scored as 0-4+, with 4+ indicating maximal activity and
0+ indicating no activity. B,
[S]Met/Cys-labeled rGal-1 and C2SrGal-1 were
applied to columns of either asialofetuin-Sepharose or
laminin-Sepharose and eluted daily with reducing or nonreducing buffer
over a period of 10 days, as described under ``Experimental
Procedures'' and as discussed in the text. The percent of
radiolabeled lectin remaining bound at each time point is indicated.
, rGal-1 bound to asialofetuin-Sepharose in reducing buffer;
, rGal-1 bound to asialofetuin-Sepharose in nonreducing buffer;
, rGal-1 bound to laminin-Sepharose in nonreducing buffer;
, C2SrGal-1 bound to asialofetuin-Sepharose in nonreducing
buffer.
We then explored whether binding of the lectin to a glycoconjugate ligand could stabilize activity of the lectin. To test this possibility, 50 µg of rGal-1 was bound to a 1-ml column of asialofetuin-Sepharose containing 5 mg/ml ligand in either the presence or absence of 2-mercaptoethanol. The column was washed with buffer either with or without 2-mercaptoethanol. This column was washed every 24 h to remove lectin that had become inactive. The rGal-1 washed from the column without hapten was collected and analyzed by SDS-PAGE. At the end of the experiment, the column was eluted with buffer containing 100 mM lactose, to estimate the amount of residual lectin left on the support. When the rGal-1 was bound to a column of asialofetuin-Sepharose, approximately one-half of the lectin appeared to retain its activity for 150 h in the absence of 2-mercaptoethanol (data not shown). In the presence of 2-mercaptoethanol, the vast majority of rGal-1 appeared to retain its activity during the 150 h (data not shown).
To directly quantify the
stability of the lectin when bound to columns of immobilized ligands,
[S]Met-labeled rGal-1 and C2SrGal-1 were
prepared by metabolic radiolabeling of E. coli expressing the
recombinant proteins. The radiolabeled lectins were bound to a column
of asialofetuin-Sepharose with or without 2-mercaptoethanol, and, every
24 h, the column was washed with either reducing or nonreducing buffer
to remove lectin that had become inactive. Fifty percent of the
[
S]rGal-1 remained bound at 5 days to the
asialofetuin-Sepharose in the absence of reducing agent (Fig. 3B). In the presence of reducing agent, more than
80% of the lectin was still bound to the asialofetuin-Sepharose column (Fig. 3B). We then tested whether a more active ligand,
the basement membrane glycoprotein laminin, could stabilize the lectin
even further. Virtually all the rGal-1 remained bound to the column of
laminin-Sepharose in the absence of reducing agent, and the t
was estimated to be >2 weeks (Fig. 3B). This may reflect the higher affinity
exhibited by galectin-1 for laminin in comparison to asialofetuin.
Laminin contains poly-N-acetyllactosamine, a high affinity
determinant for galectin-1 binding, whereas asialofetuin lacks this
structural feature and binds relatively poorly to galectin-1 (Zhou and
Cummings, 1990).
In contrast to rGal-1, the hemagglutinating
activity of C2SrGal-1 was stable when the lectin was stored in solution
in the absence of reducing agent for 48 h (data not shown). In
addition, more than 80% of the [S]Met-labeled
C2SrGal-1 in nonreducing solutions remained firmly bound to a column of
asialofetuin-Sepharose for up to 10 days (Fig. 3B).
These results demonstrate that cysteine at position 2 in the hamster
galectin-1, as also observed for the human and bovine galectin-1, is
critical in causing instability of the lectin in solutions lacking
reducing agents. It has been observed by Liao et al.(1994), in
a crystal structure of bovine galectin-1, that this Cys-2 is
disordered, whereas the other 5 cysteine residues are either buried in
the molecule or oxidized and solvated. Taken together, the results
demonstrate that galectin-1 is inactive in the absence of reducing
agents, but, more importantly, they show that reducing conditions are
not required to maintain activity of the lectin in vitro when
it is associated with high affinity glycoconjugate ligands.
Figure 4: Kinetics of binding of C2SrGal-1 to immobilized laminin. A, 5 µg of C2SrGal-1 was applied per microtiter plate well precoated with laminin (500 ng/well) and incubated for different times, as indicated. The amount of bound lectin was determined using antibody to the lectin in an ELISA-type format, as described under ``Experimental Procedures.'' Lactose (25 mM final concentration) was added after 360 min to dissociate bound lectin. In B, microtiter plate wells were coated with laminin (500 ng/well), and different amounts of C2SrGal-1 were added and incubated at 4 °C for either 2 or 20 h, as indicated. Each point represents an average of triplicate wells.
To directly investigate the possibility of monomer and dimer equilibrium of galectin-1, we used high performance size exclusion chromatography to analyze rGal-1 or C2SrGal-1 serially diluted in buffer with or without reducing agent. The diluted samples were kept at 4 °C for 24 h to promote equilibrium, and each sample was analyzed by size exclusion HPLC in a 15-min run. C2SrGal-1 was recovered as two peaks in this column with the first peak eluting at 7 min 30-45 s and the second peak at 8 min 35-50 s (Fig. 5). A number of standard proteins ranging in size from 13.7 to 150 kDa were analyzed to standardize the size exclusion properties of the TSK column. Based on the elution positions of these molecular mass standards, we estimated that the first eluted peak of C2SrGal-1 represented a 30.5-kDa protein and the later peak a 14.9-kDa protein (Fig. 5, inset). These correspond to the predicted sizes of the dimer and monomer forms of C2SrGal-1, respectively.
Figure 5:
Monomer and dimer forms of galectin-1 in
solution. A 100-µl sample of C2SrGal-1 containing 10 µg of
lectin was analyzed by size exclusion HPLC through a SW 2000 column,
and elution was monitored by A (OD214).
The column was calibrated with molecular mass markers as indicated in
the inset (bovine
-globulin, 158 kDa; bovine serum
albumin, 67 kDa; chicken ovalbumin, 44 kDa; equine myoglobin, 25 kDa;
chymotrypsinogen, 17 kDa; ribonuclease A, 13.7
kDa).
The formation of monomers and dimers was
totally dependent on the concentration of C2SrGal-1. Most of the
C2SrGal-1 existed as a dimer in the undiluted stock solution (protein
concentration of 80 µM) (Fig. 6A). In
contrast, at low concentration (80 nM) the monomeric form was
dominant. The amount of the monomeric form of the protein at different
concentrations of C2SrGal-1 was determined, the chromatograms are shown
in Fig. 6A, and a compilation of the results are shown
in Fig. 6B. It can be estimated form these data that
the apparent K of dissociation of the dimer to
monomer is
7 µM.
Figure 6:
Size
exclusion HPLC and concentration-dependent dimerization of the
C2SrGal-1. A, C2SrGal-1 (80 µM) was diluted in
PBS-azide to various concentrations and allowed to sit at 4 °C for
20 h to equilibrate. From each sample, 100 µl was analyzed by size
exclusion HPLC, as in Fig. 5. At high concentrations, A was used for protein determination, and, at
low protein concentrations, A
was used. B, the peak area was integrated, and the concentration
dependence of the monomer formation was
plotted.
Figure 7:
Time-dependent monomer formation of the
C2SrGal-1 dimers. C2SrGal-1 was diluted to 0.8 µM in
PBS-azide, size exclusion HPLC was performed following different times
of incubation, as indicated, and A was
monitored.
Figure 8:
Time- and concentration-dependent monomer
formation of rGal-1 dimers in reducing buffers. The monomer formation
of rGal-1 in reducing buffer was analyzed by size exclusion HPLC by
varying the concentration of lectin and incubation times, as indicated,
and A was monitored.
Figure 9:
Reversibility of the monomer-dimer
equilibrium. A, 100 µl of a 1/100 dilution of
[S]Met-labeled C2SrGal-1 (10 µg/ml) was
applied to the size exclusion HPLC column, and fractions were
collected. The fractions were monitored at A
,
and the radioactivity in each fraction was determined by liquid
scintillation counting. B, to the same diluted sample of
[
S]Met-labeled C2SrGal-1 (200 µl), 100
µl of nonlabeled C2SrGal-1 (1.1 mg/ml) was added and
incubated at 4 °C for 20 h to allow equilibration. One hundred
µl of this mixture was analyzed as in A.
We tested the possibility that haptenic sugar might affect the monomer-dimer equilibrium for the lectin. A concentrated form of C2SrGal-1 in dimer form was diluted to a low concentration to promote monomer formation and incubated with or without 0.1 M lactose. The samples were analyzed by size exclusion HPLC using PBS-azide buffer either with or without 0.1 M lactose. The monomer formation of the lectin was not affected by lactose (Fig. 10).
Figure 10: Effect of lactose on monomer-dimer equilibrium. A dilute amount of C2SrGal-1 in a predominantly monomeric form was incubated with or without 0.1 M lactose overnight at 4 °C. The samples were then analyzed by size exclusion HPLC using PBS-azide buffer either with or without 0.1 M lactose.
To analyze the monomer-dimer nature of the intracellular form of galectin-1 in Lec8 CHO cells, the cells were harvested, solubilized with 1% Triton X-100, and analyzed by size exclusion HPLC. Fractions were collected, and one-half of each fraction was transferred to a 96-well microtiter plate. Using anti-galectin-1 antibodies, each fraction was assayed for the lectin by ELISA. The results indicate that most of the detectable galectin-1 derived from Lec8 CHO cell extracts eluted from the HPLC column in the position of both monomers and dimers (Fig. 11). Each fraction was also analyzed by Western blot using antibodies to the lectin. The results showed that the major immunoreactive bands in Western blots corresponded exactly to the peaks of absorbance in the ELISA assay (data not shown). The monomer/dimer forms are unlikely to arise by interconversions occurring after cell lysis because of the slow rate observed for interconversion of monomer and dimer in the above experiments. Some of the lectin detected by the ELISA assay in Fig. 11may also be in a higher oligomeric form of unknown nature, but this possibility was not studied further at this time. Overall, the results indicate that galectin-1 in CHO cells exists as both monomer and dimer forms and that both forms are capable of binding glycoconjugates.
Figure 11: Monomer and dimer forms of galectin-1 in the cytosol of Lec8 CHO cells. Lec8 CHO cells were grown to confluence in a 100-mm dish, harvested, and solubilized with 1 ml of 1% Triton X-100/PBS-azide, and 100 µl was analyzed by size exclusion HPLC. Fractions (320 µl) were collected and 100 µl of each fraction was transferred to a 96-well microtiter plate. Galectin-1 was determined in each fraction using anti-galectin-1 antibodies in an ELISA-type format, as described under ``Experimental Procedures.''
We have discovered that galectin-1 from CHO cells is a monomeric protein that is able to form dimers in the micromolar range. The monomer-dimer equilibrium is both timedependent and reversible, and both forms of the lectin are able to bind carbohydrate. Lectin derived from the cytoplasm of CHO cells occurs in both monomer and dimer forms. Furthermore, we have found that the lectin is extremely stable in the absence of reducing agents when the protein is bound to carbohydrate ligands. These results are meaningful toward our understanding of this widely distributed lectin and provide new information that extends the findings of other groups.
Galectin-1 was first identified by its hemagglutinating activity and inhibition of agglutination by lactose, which indicated that the lectin is multivalent and binds to sugars on cell surfaces (Teichberg et al., 1975; de Waard et al., 1976). Two unusual features of the lectin were also found. The lectin required reducing conditions to maintain its activity, and much of the lectin was found to be intracellular (Briles et al., 1979; Barondes, 1984). Recent crystallographic analysis of bovine galectin-1 are consistent with other studies in demonstrating that the lectin is a homodimer with each subunit possessing one sugar binding site (Liao et al., 1994).
Galectin-1 from CHO cells, as from many other sources, contains 6 cysteine residues, and, in all cases examined, these occur as free sulfhydryls (Hirabayashi and Kasai, 1993). Selective mutagenesis of the cysteine residues in human galectin-1 has demonstrated that cysteine is not required for carbohydrate binding activity (Hirabayashi and Kasai, 1991; Abbott and Feizi, 1991). Rather, in the absence of reducing agent, the free cysteine residues lead to oxidative damage to the protein and perhaps to aberrant oligomerization by disulfide bond formation (Tracey et al., 1992). This requirement for reducing conditions to maintain activity led to the earlier description of galectins as sulfhydryl-type or S-type lectins. Another unusual feature of galectin-1 is that it lacks a typical secretory signal sequence and it is synthesized on free polysomes in the cytosol (Clerch et al., 1988; Wilson et al., 1989), and it is not secreted by the normal secretory pathway (Cooper and Barondes, 1990).
In terms of complex carbohydrate
binding activity, galectin-1 binds to desialylated glycoconjugates
containing clustered glycosides with terminal 1,4-linked
galactosyl residues and binds to N-acetyllactosamine better
than to lactose (Briles et al., 1977; Barondes, 1984; Lee et al., 1990). The lectin also binds with high affinity to
sialylated poly-N-acetyllactosamine sequences
[3Gal
1-4GlcNAc
1]
of the type
found in laminin and lysosome-associated membrane proteins 1 and 2
(Leffler and Barondes, 1986; Merkle and Cummings, 1988; Sparrow et
al., 1987; Do, et al., 1990; Zhou and Cummings, 1993).
These observations taken together raise several interesting and complex questions about galectin-1. How could galectin-1 be efficient and functional in binding extracellular glycoconjugates if the protein requires reducing conditions to maintain activity? Does the lectin occur in a monomeric form and how is formation of the dimer regulated? What is the nature of the intracellular and extracellular forms of the protein? Is the stability of the lectin affected by its interaction with glycoconjugates? In our study we attempted to address both the mechanisms of how the protein becomes a dimer and the significance of reducing activity to maintain lectin activity.
Our results
demonstrate that the binding of galectin-1 to appropriate
glycoconjugates can greatly stabilize the lectin in the absence of a
reducing environment. Moreover, the inactivation of the lectin not
associated with ligand could provide a type of control mechanism to
regulate the extracellular levels of the lectin in vivo and
turnover the lectin. Conceivably, the regulation of galectin-1 activity
by extracellular oxidation might be comparable to regulation of
-1-antitrypsin activity by its sensitivity to oxidation (Travis
and Salvesen, 1983). There is a report that the C-terminal half-domain
of galectin-3, which is homologous to galectin-1, is sufficient for
saccharide binding (Agrwal et al., 1993). Furthermore, this
folded polypeptide was resistant to thermal denaturation when it was
bound to lactose. If galectin-1 is bound to a proper ligand i.e. poly-N-acetyllactosamine, the correct conformation of
galectin-1 might be retained, which might prevent galectin-1
inactivation due to formation of intra- or intermolecular disulfide
bonds.
The existence of galectin-1 as a homodimer is well
established by many studies (Lobsanov et al., 1993; Liao et al., 1994), but the details of dimerization and its
significance have not been clearly studied (Roff and Wang, 1983; Beyer et al., 1980). Our results demonstrate that dimerization is
dependent on the concentration of lectin and the K for the monomer-dimer is
7 µM. In addition, this
equilibrium is fully reversible and takes about 20 h to reach. It is
interesting that the monomer-dimer equilibrium is reached at a
relatively much slower rate than the binding kinetics of the lectin to
laminin. In ELISA-type binding studies to laminin performed in a short
time assay (2 h), most of the galectin-1 is in the form of a dimer,
whereas in the longer assays (20 h), the protein is mostly monomeric.
Although we do not have a precise explanation at this point, it seems
likely that the anomalous binding behavior of the lectin observed in Fig. 4B is due to this monomer-dimer equilibrium.
Simple ligands such as lactose clearly do not affect the monomer-dimer
equilibrium. It is conceivable that glycoprotein ligands could alter
the equilibrium between monomer and dimer, but this has not yet been
investigated.
It has been reported by Wells and Mallucci(1991) that galectin-1 is an autocrine negative growth factor at concentrations in the subnanomolar range. At this concentration, the lectin would be predicted to be monomeric and raises the question of whether the lectin has a different biological function in the monomeric state than in the dimeric state. This possibility remains to be explored. One recent report suggested that galectin-1 can form an inactive tetramer at a subnanomolar concentration (Wells and Mallucci, 1992). In our study we could not detect such a tetramer; however, we could see a large aggregation product when we incubated rGal-1 (but not C2SrGal-1) in the absence of reducing agent for 30-40 h. The lectin in this aggregate was unable to bind laminin (data not shown).
In the accompanying report (Cho and Cummings, 1995), we describe our studies on the biosynthesis of galectin-1 in CHO cells. Our results reveal that the lectin secreted by cells is found both at the cell surface, where it is bound to surface glycoconjugates, and in the media in free form. As anticipated by the findings of the current study, the free form accumulating in the media of the cells is inactive whereas the form on the cell surface and in the cytoplasm of cells is functional. These studies reveal that galectin-1 has a complex regulation, involving equilibrium between monomer (non-cross-linking form) and dimer (cell-agglutinable form), inactive forms, and active forms bound to glycoconjugates. The overall biosynthesis and fate of galectin-1 in the extracellular space during biosynthesis is considered in more detail in the accompanying manuscript (Cho and Cummings, 1995).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M96676[GenBank].