(Received for publication, April 26, 1995)
From the
The Bacillus thuringiensis CryIA(c) insecticidal
The target of insecticidal Bacillus thuringiensis crystal We recently reported the
purification of this 120-kDa putative receptor from M. sexta midgut membranes by a combination of protoxin affinity
chromatography and anion-exchange chromatography(5) .
N-terminal and internal partial amino acid sequences were similar to
sequences of the ectoenzyme aminopeptidase N, and the purified 120-kDa
glycoprotein displayed aminopeptidase N but not alkaline phosphatase
activity. CryIA(c) toxin itself had no apparent effect on
aminopeptidase activity over a range of concentrations. In ligand
blotting experiments, the purified glycoprotein had the characteristics
predicted of the receptor; it bound CryIA(c) toxin in the presence of
GlcNAc but not GalNAc, it bound the lectin SBA, but it did not bind
CryIB toxin ( (5) and references therein). The same
glycoprotein was partially purified by Sangadala et al.(6) who used isoelectric focusing and immunoaffinity
chromatography to obtain a mixture of 120- and 65-kDa midgut brush
border proteins from M. sexta. Both (glyco)proteins bound
CryIA(c) toxin in ligand blots, although the 120-kDa band was the major
toxin-binding component(4) . Enzyme assays revealed both
aminopeptidase and alkaline phosphatase activity in the partially
purified preparation, and the 120-kDa protein was identified as
aminopeptidase N from the partial amino acid sequence. When
reconstituted into phospholipid vesicles, the protein mixture increased
toxin binding by 35% and enhanced toxin-induced Aminopeptidase N (CD13; microsomal aminopeptidase;
Following receptor binding at
the midgut epithelium, toxins probably act by opening nonspecific
channels or pores in the membrane, which leads to colloid osmotic lysis
of midgut cells and ultimately the death of the insect(11) .
With the aim of understanding both the biochemical basis for toxin
specificity and the mechanism(s) by which membrane insertion and
cytolysis occur, we have cloned and sequenced the cDNA of M. sexta aminopeptidase N, a putative CryIA(c) receptor.
Figure 2:
Sequence of M. sexta aminopeptidase N cDNA and deduced amino acid sequence. The
putative N-terminal cleavable signal peptide is underlined,
and consensus N-linked glycosylation sites are double-underlined. Partial amino acid sequences from the
purified protein are broken underlined, and the N-terminal
residue of the mature protein determined by Edman degradation is
designated by a
Figure 1:
Aminopeptidase
clones and PCR primers. A, relationship between
A fully degenerate
antisense reverse PCR primer 3R was designed from internal amino acid
sequence 68.5, QIVDDVF(5) . This primer was used in conjunction
with forward primer 3F to amplify fragments of aminopeptidase N cDNA
from M. sexta midgut single-stranded cDNA preparations (Fig. 1). A single 1700-bp PCR product was identified by
hybridization with 4F and was gel-purified and directly sequenced. This
unambiguous gene sequence was used to design a unique reverse PCR
primer 5R (see Fig. 1), situated 345 bp downstream of the unique
forward PCR primer 3F.
The 2970-bp open reading frame encodes a protein of 990 residues (Fig. 2). The N-terminal sequence of the mature (purified)
protein determined by Edman degradation extends from Asp
Figure 3:
Aminopeptidase sequence alignment.
Alignment of the deduced amino acid sequences of aminopeptidase N from M. sexta with aminopeptidase N from human(18) ,
rabbit(19) , and rat(20) , aminopeptidase yscII from S. cerevisiae(21) , alanine aminopeptidase (pepN) from L. delbruckii(22) , and the
aminopeptidase A from human (24) and mouse(23) . Letters in the consensus sequence represent residues common to
all sequences. Numbers to the left refer to the first
residue in each line relative to the start codon of each respective
primary sequence. A, highly conserved block including the zinc
binding/catalytic site typical of the aminopeptidase family of
gluzincins. Conserved sequences are boxed. The gluzincin motif
is shown above the consensus sequence, with catalytic residues in boldface and zinc binding ligands in boldface
italics. B,M. sexta aminopeptidase N
C-terminal extension containing the GPI signal peptide not found in
other aminopeptidases.
A hydropathy plot of
the predicted primary structure (Fig. 4A) reveals one
region at the N terminus and one at the C terminus with hydropathy
averages greater than 1.6 and thus capable of spanning the membrane in
a helical conformation(28) . Although there is a third and
comparatively shorter hydrophobic region centered around
Ala
Figure 4:
A, hydropathy plot of the M.
sexta aminopeptidase N protein sequence. The method of Kyte and
Doolittle (28) was used with averaging over a window of 11
residues. Hydrophobicity resulted in positive and hydrophilicity in
negative values. B, schematic diagram of M. sexta aminopeptidase N protein sequence. The predicted N-terminal
cleavable signal peptide (openbox) is followed by a
predicted pro-peptide (filledbox). The horizontallyhatchedbox represents the
gluzincin motif, while at the C terminus there is a predicted O-glycosylated stalk (dottedbox) and the
GPI signal peptide (diagonallyhatchedbox).
The four potential N-linked glycosylation sites are indicated
as knobs.
A closer examination of the C-terminal
hydrophobic sequence suggests that it is not the stop-transfer sequence
of a type I membrane protein (27) since it lacks charged
residues flanking the hydrophobic region, particularly positive charge
at the C terminus typically found in such
sequences(32, 33, 34) . However, it does show
the characteristics of a signal peptide for the addition of a
glycosylphosphatidylinositol (GPI) anchor: a C-terminal run of 19
hydrophobic residues (Ile A
common diagnostic test for a GPI-anchored protein (37) is to
demonstrate its release from the membrane by bacterial PI-PLC.
Following incubation of M. sexta BBMV with PI-PLC, 16.0
± 0.4% of total aminopeptidase N activity was released into the
supernatant (n = 4) compared with a release of 4.8
± 0.9% in the absence of PI-PLC (n = 7). In
comparison, PI-PLC released 83.5 ± 2.6% of alkaline phosphatase
activity into the supernatant (n = 4), compared with
9.9 ± 3.2% release in the control (n = 4).
Differential solubilization by detergents can also be used to predict
the presence of a GPI membrane anchor(38, 39) , since
only detergents with a high critical micellar concentration (CMC) are
able to release significant amounts of GPI-anchored ectoenzymes into
the supernatant. Treatment of M. sexta BBMV with 0.5% CHAPS
(high CMC) released 78% of the total aminopeptidase N activity into the
supernatant (n = 2), while 0.1% Triton X-100 (low CMC)
released only 7% of total activity (n = 1). Although
PI-PLC releases only a fraction of the total aminopeptidase N activity
into the supernatant, this result demonstrates that at least a
proportion of the M. sexta enzyme is linked to the brush
border membrane by a GPI anchor. A similar study (40) showed
that aminopeptidase N in the brush border membrane of the closely
related lepidopteran Bombyx mori is also GPI-anchored. PI-PLC
caused a maximal 40% release of B. mori aminopeptidase N
activity compared with a 90% release of alkaline phosphatase activity. In this study, partial amino acid sequence from
aminopeptidase N purified from M. sexta midgut epithelium as a
putative B. thuringiensis CryIA(c) toxin receptor was used to
isolate M. sexta midgut aminopeptidase N cDNA clones by a
PCR-based approach. Analysis of the 990-residue deduced amino acid
sequence indicates that it is a large prepro-protein (Fig. 4B). The two pre-regions are the C-terminal GPI
signal sequence (residues 968-990) and the (predicted) N-terminal
cleavable signal sequence (residues 1-15), while the sequence
between the predicted signal peptidase cleavage site and the N terminus
of the mature protein (residues 16-35) is presumably a
pro-region. Following proteolytic release of these pre- and
pro-sequences, the mature polypeptide would then be 934 residues long,
with a calculated molecular mass of 105 kDa. A 33-amino-acid long
region (residues 935-967) immediately preceding the GPI signal
peptide is rich in serine and threonine residues, which are potential O-glycosylation sites, and also in the helix-breaking amino
acid proline, commonly found in A number of ectoenzymes are now known to
possess GPI membrane anchors including acetylcholinesterase, alkaline
phosphatase, microsomal dipeptidase, 5`-nucleotidase, trehalase, and
aminopeptidase P in mammals (reviewed in (42) and (43) ) and alkaline phosphatase (44) and aminopeptidase
N (40) in the midgut of the lepidopteran larva B.
mori. It is common to find that treatment of ectoenzymes with
PI-PLC releases only a fraction of the total activity. This observation
implies that the uncleaved enzyme population is either anchored by a
modified GPI structure that is insensitive to PI-PLC (reviewed in (36) ) or by a conventional C-terminal hydrophobic amino acid
sequence that arises by alternative splicing of a single mRNA
transcript, as is known to be the case with neural cell adhesion
molecules(46) . Although M. sexta aminopeptidase N
activity is relatively resistant to PI-PLC release, this latter
explanation seems unlikely since Northern blot analysis indicates that
there is only one aminopeptidase N transcript in M. sexta midgut mRNA preparations. In addition to its role as a receptor for B.
thuringiensis CryIA(c) toxin, aminopeptidase N is known to be
commandeered as a receptor by human (9) and porcine (8) coronaviruses, and by a human herpesvirus(10) . In
the latter two cases, studies demonstrated that the catalytic site and
the viral binding site were on different domains and that
aminopeptidase enzyme activity was not necessary for viral
infection(10, 47) . In our hands CryIA(c) toxin has no
effect upon aminopeptidase N activity, which suggests that, like the
viruses, the toxin binds at a site distinct from the catalytic site. As
an exopeptidase, aminopeptidase N cannot itself be involved in the
proteolytic activation (48, 49) of the 133-kDa
CryIA(c) protoxin to the 66-kDa active toxin. Nor could it be
responsible for cleavage of loop regions within the active
toxin(50) , although in theory the enzyme could contribute to
any N-terminal trimming reactions following endoprotease cleavage.
Thus, it seems that the feature of aminopeptidase N being exploited by
both the viruses and CryIA(c) is simply its abundance at the apical
membrane of epithelial cells, irrespective of its function as a
protease. This does not preclude the possibility that following binding
to the aminopeptidase receptor, CryIA(c) toxin may subsequently
interact with other membrane components to which aminopeptidase N is
functionally linked. Vadlamudi et al.(51) identified (and subsequently purified) a 210-kDa
putative CryIA(b) receptor in the brush border membrane of M.
sexta. The same authors (45) recently reported the cloning
from M. sexta of this putative CryIA(b) receptor. The cDNA
clone encodes a novel cadherin-like glycoprotein which, when expressed
in either COS-7 or human embryonic 293 cells, was able to bind CryIA(b)
toxin in ligand blotting experiments and in the latter case also in
homologous binding assays. The demonstration (6) that
partially purified aminopeptidase, when incorporated in liposomes,
requires dramatically less CryIA(c) toxin to induce a given amount of The nucleotide
sequence reported in this paper has been submitted to the
GenBank
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-endotoxin binds to a 120-kDa glycoprotein receptor in the larval
midgut epithelia of the susceptible insect Manduca sexta. This
glycoprotein has recently been purified and identified as
aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were
isolated, and their combined 3095-nucleotide sequence contains an open
reading frame encoding a 990-residue prepro-protein. The N-terminal
amino acid sequence derived from the glycoprotein is present in the
open reading frame, immediately following a predicted cleavable signal
peptide and a pro-peptide. There are four potential N-linked
glycosylation sites. The C-terminal sequence contains a possible
glycosylphosphatidylinositol (GPI) anchor signal peptide, which
suggests that, unlike most other characterized aminopeptidases, the
lepidopteran enzyme is anchored in the membrane by a GPI anchor. This
was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by
phosphatidylinositol-specific phospholipase C. The deduced amino acid
sequence shows significant similarity to the zinc-dependent
aminopeptidase gene family, particularly in the region surrounding the
consensus zinc-binding motif characteristic of these enzymes.
-endotoxins is the apical (brush border) membrane of
larval midgut cells(1) . In vitro binding assays have
demonstrated that the CryIA(c) toxin binds specifically and with high
affinity to a single receptor species in brush border membranes
prepared from larvae of the susceptible lepidopteran, Manduca
sexta(2) . Ligand blotting experiments have identified a
single 120-kDa toxin-binding glycoprotein in M. sexta larval
midgut membranes as the most likely candidate for the cellular CryIA(c)
receptor(3, 4) .
Rb
release up to 1000-fold. This
important result is the first (and so far only) demonstration that a
partially purified receptor can potentiate the action of a toxin in
vitro.
-aminoacyl-peptide hydrolase (microsomal); EC 3.4.11.2) is a well
documented zinc-dependent peptidase that catalyzes removal of
N-terminal, preferentially neutral residues from peptides (reviewed in (7) ). This ectoenzyme is commonly found in the brush border
membranes of the alimentary tract in a variety of different organisms.
Recent reports have shown that a number of coronaviruses and a
herpesvirus use aminopeptidase N as a receptor in their target tissue (8, 9, 10) .
Polymerase Chain Reaction
Amplification
PCRs(
)were performed by
standard techniques(12) . If the PCR product was to be
sequenced, Pfu DNA polymerase (Stratagene) was used in the
amplification because of its high fidelity. Otherwise, Taq DNA
Polymerase (Promega) was used in all PCRs. Single-stranded cDNA from M. sexta midgut brush border membranes was prepared as
described previously(5) .
Oligonucleotide Synthesis and
Labeling
Oligonucleotides used as PCR primers and hybridization
probes were synthesized on a Millipore Expedite 8909 nucleic acid
synthesizer. Oligonucleotide probes were 3` end-labeled with
digoxigenin using the digoxigenin oligonucleotide tailing kit from
Boehringer Mannheim and were used according to the manufacturer's
recommendations.cDNA Cloning and Sequencing
The M. sexta midgut brush border membrane cDNA library in gt10 was a gift
from Dr. J. Van Rie, Plant Genetic Systems, Belgium. The library was
screened by the PCR-based microtiter plate technique described by
Israel(13) . Briefly, 8000 pfu arranged at 125 pfu/well in an 8
8 well array were screened by PCR (primers 3F and 5R). Two
wells tested positive, and the phage from these were titered and
rescreened at 4 pfu/well. One PCR-positive well from the secondary
screen was selected, individual phage clones were plaque-purified, and
phage DNA was prepared by the plate lysis method(12) . Their
identity as aminopeptidase N clones was confirmed by Southern blotting.
Subcloning and DNA Sequence Analysis
The APN
cDNA insert was excised from the phage vector with EcoRI and
subcloned into EcoRI-cut pBluescript II SK(-)
(Stratagene). The
5`APN blunt-ended PCR product was subcloned into EcoRV-cut pBluescript II SK(-), and the phage vector
sequence was excised on a BamHI fragment. All subcloning
operations were performed by standard techniques(12) . DNA was
sequenced on an Applied Biosystems Inc. 373 automated DNA sequencer,
using an Applied Biosystems Inc. Dye-Deoxy Terminator Cycle sequencing
kit. A double-stranded nested deletion kit (Pharmacia Biotech Inc.) was
used to generate a set of progressively smaller subclones of
APN
for sequencing. All clones were completely sequenced on both strands.
DNA and protein sequences were assembled and analyzed using the
Genetics Computer Group program package (14) and the Lasergene
package (DNASTAR).
PI-PLC Digestions and Aminopeptidase N
Assay
M. sexta brush border membrane vesicles, prepared
as described(15) , were suspended at 2 mg/ml in
phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl,
10 mM NaHPO
, pH 7.4). PI-PLC from Bacillus cereus (Sigma) was added at a final concentration of
2 units/ml and incubated for 90 min at 30 °C. The vesicles were
pelleted by centrifugation at 13,000
g for 10
min, the pellet resuspended in the same volume of phosphate-buffered
saline, and the supernatant and pellet assayed for aminopeptidase and
alkaline phosphatase activity as described(5) . Control release
was measured under the same conditions in the absence of PI-PLC.
Release by detergents was carried out by the same method, using final
concentrations of 0.1% (v/v) Triton X-100 or 0.5% (w/v) CHAPS.
Partial Amino Acid Sequence and PCR
Following
purification of aminopeptidase N from M. sexta midgut
epithelium, both N-terminal and internal partial amino acid sequences
were obtained from the glycoprotein. A possible overlap between the
N-terminal sequence and internal amino acid sequence 77 (5) was
tested by nested PCR using fully degenerate primers. When subcloned and
sequenced, the cDNA sequence confirmed the overlap between the two
partial amino acid sequences and also yielded 45 bp of unambiguous
aminopeptidase N gene sequence (nt 139-183 in Fig. 2),
which was used to design a unique forward PCR primer 3F and an
oligonucleotide probe 4F (Fig. 1).
. The zinc binding/catalytic site (gluzincin
motif) is boxed. The GPI signal peptide is dot-underlined, and
indicates the probable
cleavage/attachment site of the anchor moiety. Repeats in the
3`-untranslated tail are underlined, and the two
polyadenylation signals are in capitals.
5`APN (top) and
APN (bottom) and location of
oligonucleotides used as PCR primers (arrows) and
hybridization probes (lines). Primer 3R (brokenline) is a fully degenerate oligonucleotide predicted
from partial amino acid sequence, while all other oligonucleotides are
designed from a unique cDNA sequence. The scale refers to the position
in the combined cDNA sequence (Fig. 2). B, sequence of
unique oligonucleotides used in PCR
amplifications.
Isolation of Two Overlapping Clones for Aminopeptidase
N
The unique primer pair 3F/5R was used to screen a M. sexta midgut cDNA library in gt10 using the high stringency
PCR-based technique of Israel(13) . 8000 phage clones were
screened, and one positive recombinant phage,
APN, was obtained.
Although the 2994-bp cDNA insert (nt 102-3095 in Fig. 2) was
found to contain an open reading frame that encoded the N terminus and
all eight tryptic peptides derived from the purified
protein(5) , no initiating ATG codon was found, indicating that
clone
APN does not contain the total mRNA sequence. Attempts to
obtain the missing 5` end of the mRNA by 5`-rapid amplification of cDNA
ends (16) were unsuccessful, and therefore the cDNA library was
screened again by nested PCR, using a forward primer (gtLF) sited in
gt10 and two nested reverse primers (8R and 9R) at the 5` end of
clone
APN (see Fig. 1). A single 350-bp PCR product,
5`APN, was obtained containing 148 bp of aminopeptidase N cDNA (nt
1-148 in Fig. 2), including a 47-bp overlap with the 5`
end of
APN. The new 5` cDNA still did not contain an initiating
ATG codon, but it did encode a putative N-terminal cleavable signal
peptide (see below).
Nucleotide and Deduced Amino Acid Sequence
Both
APN and
5`APN cDNAs were subcloned and sequenced on both DNA
strands as described under ``Experimental Procedures.'' The
combined 3095-bp nucleotide sequence (Fig. 2) has an in-frame
ATG codon at the 5` end of the cDNA (nt 94-96), but this is
probably not a start codon since it does not meet the criteria for a
Kozak consensus translational initiation site(17) . Therefore,
the combined cDNA sequence is presumed to be missing a 5` upstream
sequence, including the initiating ATG codon. There is a long open
reading frame starting at nucleotide 2 and extending 2970 bp to a TAA
stop codon at nucleotide 2971. The short 124-bp 3` noncoding region
includes two additional in-frame stop codons and two consensus AATAAA
polyadenylation signals contained within a 17-bp repeat (nt
2989-3006 and nt 3064-3081, Fig. 2), which may imply
the occurrence of polymorphism in the 3` noncoding region of the mRNA.
to Pro
(Fig. 2). The presence of residues
upstream of Asp
suggests that in M. sexta,
aminopeptidase N is synthesized as a larger precursor protein and is
trimmed to a mature product by limited proteolysis. There are four
consensus Asn-X-Ser/Thr sequences, indicating possible N-glycosylation sites in the protein.
Homology to Other Aminopeptidases
Searches of the
SwissProt (EMBL) protein sequence data base with the primary structure
of M. sexta aminopeptidase N showed significant similarity to
human (31% identity)(18) , rabbit (31% identity)(19) ,
and rat aminopeptidase N (31% identity)(20) , to aminopeptidase
yscII from Saccharomyces cerevisiae (29%
identity)(21) , alanine aminopeptidase (pepN) from
lactobacterium (27% identity) (22) , and also to mouse (29%
identity) (23) and human (28% identity) (24) aminopeptidase A. A multiple sequence alignment between
these known aminopeptidase sequences and the M. sexta sequence
showed that the most striking similarity was around the characteristic
and functionally crucial zinc-binding motif between residues
Ile and Phe
(Fig. 3A). This
sequence classifies the M. sexta protein as a member of the
aminopeptidase family of gluzincins, with His
,
His
, and Glu
being zinc ligands and
Glu
being involved in catalysis(25) . An obvious
difference in the alignment was the C-terminal 40-60-residue
extension of the M. sexta sequence, which includes the GPI
anchor signal peptide (Fig. 3B; see below). This
feature probably reflects the fact that other membrane-bound
aminopeptidases are generally anchored by an N-terminal signal anchor
sequence.
Membrane Anchoring
In the epithelial cells of
mammalian kidney and intestine aminopeptidase N is a type II membrane
protein, anchored by an uncleaved N-terminal signal anchor sequence and
with a C-terminal extracellular domain(26, 27) .
However, treatment of M. sexta brush border membrane vesicles
(BBMV) with proteinase K leads to the release into the supernatant of a
100-kDa soluble form of aminopeptidase N, with the same N-terminal
amino acid sequence as the membrane-bound form of the protein (data not
shown). This suggests that the M. sexta aminopeptidase N is a
type I membrane protein, anchored in the membrane by a C-terminal
``stop-transfer'' sequence and with an N-terminal
extracellular domain. Such a topology would require an N-terminal
cleavable signal peptide to initiate translocation across the
endoplasmic reticulum membrane(27) ., biochemical analysis of the protein (preceding
paragraph) indicates that it is unlikely to be a transmembrane helix.
Analysis of the N-terminal hydrophobic region by the weight-matrix
method of von Heijne (29) for predicting signal sequence
cleavage sites yields a ``score'' of 7.8 for cleavage after
residue 15, while all other residues give scores of 1.9 or less. Known
cleavage sites in other proteins typically have scores of 6-12.
The algorithm gives a correct prediction in 75-80% of
cases(29) , and on this evidence it seems probable that the N
terminus of the deduced polypeptide is a cleavable signal sequence,
with scission by the signal peptidase occurring on the C-terminal side
of Thr
. According to this predicted topology, the sequence
between Thr
and Asp
(the N terminus of the
mature protein as determined by Edman degradation) constitutes a
propeptide, the proteolytic release of which might serve to activate
pro-aminopeptidase N. Although activation propeptides are a common
feature of many proteases, hormones, and growth factors (reviewed in (30) ), there is only one other example of a putative
propeptide region in an aminopeptidase, predicted from the cDNA-derived
primary structure of aminopeptidase Y in S.
cerevisiae(31) .
-Ala
),
preceded by a cluster of three small residues
(Gly
-Gly
, see Fig. 2), which
functions as a cleavage/attachment site(35, 36) .
-turns. By analogy to decay
accelerating factor, sucrase/isomaltase, low density lipoprotein
receptor, and the mucin protein family (reviewed in (41) ),
this region may represent a rigid, O-glycosylated stalk that
serves to elevate the active site of the enzyme well above the cell
surface. The mature protein sequence also has four consensus N-glycosylation sites, and lectin binding studies have
indicated that at least one of these sites is occupied.
(
)The presence of covalently attached carbohydrate may
explain the observed difference between the molecular mass of the
purified enzyme (120 kDa) and that of the polypeptide predicted from
cDNA sequence (105 kDa).
(
)Therefore the
relative resistance of M. sexta aminopeptidase N to PI-PLC
cleavage is probably due to modification to the GPI anchor structure
itself.
Rb
leakage compared with vesicles
containing no brush border membrane proteins strongly suggests that the
120-kDa aminopeptidase N glycoprotein functions as a CryIA(c) receptor in vivo. Having cloned M. sexta aminopeptidase N, we
are in a position to directly investigate its interaction with CryIA(c)
toxin.
/EMBL Data Bank with accession number
X89081[GenBank Link].
We acknowledge the assistance of Drs. L. Packman and
C. Hill for oligonucleotide synthesis, supported by the Wellcome Trust,
and P. Barker at the Biotechnology and Biological Sciences Research
Council, Babraham Institute for automated DNA sequence analysis. We
gratefully acknowledge Dr. J. Van Rie for the gift of the gt10
cDNA library, which was used to complete the PCR-derived cDNA sequence.
We thank Dr. N. Crickmore and T. Sawyer for technical assistance, and
Dr. C. E. Cummings for reviewing the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.