Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 481-487
A Novel Asn344 Deletion in the Core Domain of Coagulation Factor
XIII A Subunit: Its Effects on Protein Structure and Function
By
Sasichai Kangsadalampai,
Gareth Chelvanayagam,
Rohan T. Baker,
Pa-thai Yenchitsomanus,
Parichat Pung-amritt,
Chularatana Mahasandana, and
Philip G. Board
From the Molecular Genetics Group, John Curtin School of Medical
Research, Australian National University, Canberra, Australia; the
Medical Molecular Biology Unit, Office for Research and Development,
Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok; and
the Department of Paediatrics, Faculty of Medicine Siriraj Hospital,
Mahidol University, Bangkok, Thailand.
 |
ABSTRACT |
In this study a previously undescribed 3 bp deletion,
AAT1030-1032, in the factor XIII A subunit gene,
has been detected in a Thai patient. The inframe deletion results in
the translation of a factor XIII A subunit that lacks Asn344. This is
the first inframe deletion to be identified in the factor XIII A
subunit gene because six previously reported deletions have all caused
frameshifts. The deletion has been introduced into a factor XIII A
subunit cDNA and the deleted polypeptide expressed in yeast. The mRNA encoding the mutant enzyme appears to have normal stability but the
translated protein is subject to premature degradation. In addition,
the mutated enzyme exhibited very little transglutaminase activity
compared with the wild-type enzyme. Structural modeling of the deleted
enzyme suggests that the absence of Asn344 would have a potent impact
on the catalytic activity by reorienting the residues associated with
the catalytic center. Thus, the Asn344 deletion strongly confirms the
significance of the residues surrounding the catalytic center of the
factor XIII A subunit.
| |
INTRODUCTION |
BLOOD COAGULATION factor XIII is a
proenzyme for a plasma transglutaminase previously known as fibrin
stabilizing factor. It is involved in the modification of fibrin clots
by the formation of tight covalent cross-links between fibrin monomers
or between fibrin and 
-2 plasmin inhibitor.1 This
cross-linking enhances the mechanical strength of the clot and
increases its resistance to proteolysis. Factor XIII has also been
found in many cells and tissues (platelets, megakaryocytes, placenta,
uterus, and monocytes/macrophages).2-7 Factor XIII
circulating in plasma is composed of two A and two B subunits forming a
noncovalent heterotetrameric complex (A2B2),
whereas the intracellular form is a dimer of A subunits
(A2).8,9 The A subunits of factor XIII are
responsible for its catalytic activity,10,11 whereas the B
subunits are thought to play a significant role as a protective carrier
of the circulating A subunits.12-14 In addition, there is
some evidence that the B subunit might regulate the activation of the
proenzyme A subunits.10,11
Inherited factor XIII deficiency can result from mutations in either
the A- or B-subunit genes, but the frequency of A-subunit mutations is
far higher than that of B-subunit mutations. This difference is
probably due to the fact that B-subunit deficiency is less severe and
possibly goes undiagnosed. Congenital factor XIII A-subunit deficiency
results in a severe life-long bleeding diathesis and frequently
presents as umbilical bleeding a few days after birth.15
Other clinical features of this disease including abnormal wound
healing and spontaneous abortion have been reviewed by Board et
al.16 Inherited factor XIII deficiency is an autosomal
recessive disorder as the genes coding for the A and B subunits have
been precisely mapped to chromosome band 6p24-2517 and
chromosome band 1q31-32.1,18 respectively.
Inherited factor XIII deficiency has been reported in many racial
groups,16 and although many mutations have been identified in the A-subunit gene, only 3 mutations have been detected in the
B-subunit gene.19,20 Since the first two mutations causing factor XIII A-subunit deficiency were reported in
1992,21,22 around 30 mutations have been
identified.23-37 It is notable that the mutations observed
in the factor XIII A-subunit gene are heterogeneous, and only four
mutations have been reported on multiple occasions (C 
T
transition at codon 661,25,35 G A transition at codon 681,21,38 AATT deletion at codons
462-46331,34). All the nucleotide deletions reported so far
have been found to disrupt the reading frame.
In this study, we identified a novel 3-bp deletion in a homozygous
patient with Thai origins. This deletion causes the translation of a
polypeptide missing Asn344. The effect of the deletion on the enzyme's
expression and activity has been investigated by site-directed
mutagenesis, and its effect on enzyme structure has been predicted by
computer modeling.
| |
MATERIALS AND METHODS |
Patient.
The patient is a Thai girl born in 1992 and the third child in the
family. According to the interview at the time of admission, her
parents are not relatives and there is no history of bleeding disorders
in either side of the family. The first child in this family died of
intracranial hemorrhage before this disorder was diagnosed in the
family. The patient's severe factor XIII deficiency first became
evident when bleeding from the umbilical stump was noted 7 days after
birth. Nineteen months later, she suffered from posttraumatic hematoma
of the scalp, and at 2 years of age she had a posttraumatic subgaleal
hematoma. Another year later, she became ill with posttraumatic
intraventricular hemorrhage. A comprehensive hematological
investigation when the patient was 3 years and 8 months showed that she
had a complete blood count within the normal range and had normal
values for basic blood coagulation and platelet function tests.
However, her fibrin clots solubilized very rapidly in 8 mol/L urea (10 minutes), confirming a clinical diagnosis of factor XIII deficiency.
The patient is not on regular substitution treatment but receives
fresh-frozen plasma when she suffers bleeding episodes after accidents.
The samples used in this study were collected at varying times after replacement therapy and could be potentially contaminated with residual
donor factor XIII.
DNA amplification, heteroduplex analysis, and nucleotide sequencing.
Each of the 15 exons of the factor XIII A-subunit gene was amplified
from genomic DNA from the patient and her immediate family members as
described by Board et al.21 The polymerase chain reaction
(PCR) products were then subjected to heteroduplex
analysis.39 Exons showing heteroduplex formation were
further purified by a Wizard PCR Preps DNA purification system
(Promega, Madison, WI) and directly sequenced using a
Thermo Sequenase cycle sequencing kit (Amersham, Arlington Heights,
IL).
Genetic analysis.
Genetic transmission of the 3-bp deletion in this particular family has
been linked to the length polymorphism of the tetranucleotide repeat
element (AAAG)n occurring upstream of the coding sequence of the factor XIII A-subunit gene as described
previously.40
Determination of factor XIII activity in plasma.
Plasma factor XIII activity of the patient and her close family members
was determined by the incorporation of [1,4-14C]
putrescine dihydrochloride into casein41 and by fibrin cross-linking in clotted plasma.36
Recombinant factor XIII A subunit.
The plasmid pGal181 was used for the expression of wild-type and Asn344
deleted A-subunit cDNAs. These constructs have a similar configuration
to that constructed by Jagadeeswaran and Haas.42 The
expression of the factor XIII A subunit is under the control of the
GAL1 promoter and can be induced by galactose.
In vitro mutagenesis.
The deletion of AAT at codon 344 was introduced into the factor XIII
A-subunit cDNA by the primer (5
-CAAATTGGCATCATCATGGGC-3) and the
oligonucleotide-directed in vitro mutagenesis system version 2.1 (Amersham). A plasmid containing the desired mutant was transformed into Saccharomyces cerevisiae AH22 by the lithium
acetate method43 and grown on selective media without
leucine (SD/glucose-Leu).44
Characterization of the mutant enzyme in yeast lysate.
Yeast strains were grown overnight in YPD
broth,44 washed with sterile water, and grown for another
30 hours in SD/galactose-Leu broth.44 The yeast pellet was
resuspended in lysis buffer (50 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, 1 mmol/L phenylmethylsulphonyl fluoride)
and lysed by the method described by Bartel et al.45
Immunoblotting of the mutant protein after separation on 8% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed with rabbit anticoagulation factor XIII A-subunit serum
(Calbiochem, San Diego, CA) as previously described.46 The transglutaminase activity of the mutant
enzyme was determined by the incorporation of [1,4-14C]
putrescine dihydrochloride (Amersham) into casein (Sigma, St Louis,
MO).41 A pulse-chase experiment was
performed47 using 35S-Met (Amersham) to
evaluate the possible degradation of mutant A subunits expressed in
yeast.
RNA hybridization analysis.
Because no suitable samples from the patient and her family were
available for mRNA analysis, mRNA was evaluated in yeast expressing the
mutant enzyme. Yeast strains were grown overnight in SD/glucose-Leu,
washed with sterile water, and grown for 8 hours in either YPD broth or
in YPGal broth.44 Total RNA was prepared by
the glass bead/phenol/chloroform extraction procedure,48 and aliquots (approximately 10 µg) were electrophoresed in 1% (wt/vol) agarose gel in MOPS buffer/1% (vol/vol) formaldehyde as
described by Finley et al.49 After electrophoresis, samples were transferred to a nylon membrane (Amersham Hybond N+)
as described by the manufacturer. The probe was a 2.3-kb Pst I
fragment that contained the entire factor XIII A-subunit cDNA coding
region,50 which was labeled by a random primer fluorescein labeling kit (DuPont NEN, Wilmington, DE) and hybridized
according to the manufacturer's instructions. Hybridization was
detected using CDP-Star chemiluminescence (DuPont NEN).
Computer modeling.
Computer modeling was used to examine the likely effect of the deletion
on the enzyme structure. A model for the mutant enzyme was created by
deleting Asn344 from the wild-type structure and then performing energy
minimization to resolve the excessive bond length between residues 343 and 345. The three-dimensional structure of factor XIII A subunit
determined by Yee et al51 was used as the template to model
the Asn344 deletion mutant. Modeling was performed with the Insight
package from MSI/Biosym (San Diego, CA). Initially, all atoms of
residue 344 were deleted and a pseudo peptide bond created between
residues 343 and 345. Hydrogens were introduced into the model with the
Builder module and residues 185-515, the "core" domain, were
minimized with the Discover module for 100 steps of steepest descent
minimization. In keeping with other simulations done in vacuum, a
noncovalent interaction cutoff 12 Å was used with no cross or Morse
terms. Subsequently, a short dynamics trajectory was calculated
tethering all residues but those between 340 and 349 to their original
positions with a moderate force constant of 100 kcal/mol. The system
was equilibrated for 300 iterations before calculating a single
trajectory for 500 iterations at 301°K. The resulting structure was
then further minimized for 100 steps of steepest descent minimization
followed by 200 steps of conjugate gradient minimization. The same
procedure was also applied to the wild-type core domain as a control.
The atomic coordinates of the protein structure were obtained from the
Brookhaven Protein Data Bank under the identifier
"" (Http://www.pdb.bnl.gov).
| |
RESULTS |
Plasma factor XIII activity was determined in the patient and other
members of her family. The value obtained for the patient's sample was
very low when compared with normal controls (Table 1). The values obtained from the patient's
heterozygous relatives are also low, but not lower than might be
expected given the wide normal range. An analysis of the cross-linking
in the fibrin clots of the patient and her immediate family members has
also been performed. No 
-dimer or polymer formation was observed
in the patient's fibrin clot (Fig 1). A
Western blot of the patient's plasma revealed only a trace band of
A-subunit antigen (data not shown). These data confirm the diagnosis of
A-subunit deficiency originally made after a positive urea solubility
test in the patient.
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| Fig 1.
Fibrin cross-linking in plasma clots of family members
where the Asn344 deletion was identified. (A) Fibrin clots in the
reaction containing CaCl2 and thrombin (test). (B) Fibrin
clots in the reaction containing EDTA and thrombin (control). In each
panel: lane 1, maternal sample; lane 2, paternal sample; lane 3, patient's sample; lane 4, brother's sample; and lane 5, a normal
control sample. The symbols ,  , represent uncross-linked
fibrin chains, n represents chain polymers, and
- represents chain dimers.
|
|
Heteroduplex analysis of each factor XIII A-subunit exon from the
patient indicated the presence of a deletion in exon 8 (Fig 2). Direct sequencing of amplified exon 8 DNA from the patient and her immediate family members revealed a 3-bp
(AAT) deletion of nucleotides 1030-1032 (nucleotides and amino acids
are numbered from the first serine of the mature factor XIII A-subunit
protein). This deletion does not alter the reading frame and results in a protein without Asn344 as shown in Fig 3.
There is no alteration in a restriction-cleavage site available for the
PCR-restriction fragment length polymorphism (RFLP)
diagnosis of this particular mutation; however, the mutation can be
detected directly by separation of the amplified exon 8 DNA on 6%
polyacrylamide gel since the deletion allele (208 bp) runs faster than
the normal allele (211 bp; Fig 2). The data suggest that the patient is
a homozygote having inherited the same deletion allele from both
parents. In addition, a family study using the polymorphic short tandem
repeat (AAAG)n in the 5 flanking sequence of the factor
XIII A-subunit gene (Fig 4) shows that the
patient inherited a common repeat allele from each parent, thus
supporting the conclusion that the patient is a homozygote. This study
also revealed that a deletion allele in the patient's brother, who is
heterozygous for this deletion, was inherited through the paternal
line.
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| Fig 2.
Heteroduplex analysis of amplified exon 8 DNA. The
mixture of the patient's and a normal subject's amplified DNA was run
on the first lane on the right. Amplified normal exon 8 DNA was run as
a control (second from right). The unmixed amplified DNA from the
patient, her immediate family members, and normal control were also run
on the same gel as indicated by the pedigree. The position of
heteroduplexes (between normal and 3-bp deleted strands) and the length
of homoduplexes (both normal and mutant alleles) are indicated. The
genetic transmission of the 3-bp deletion is shown. The symbols for
male ( ) and female ( ) are hatched according to the alleles
present.
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| Fig 3.
Direct sequencing of amplified exon 8 DNA. The normal and
mutated sequences are given on the left of the ladder. The missing nucleotides AAT between the positions 1030 and 1032 are shown in bold.
The asterisk (*) indicates the position of the deletion.
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| Fig 4.
The cosegregation of the 3-bp deletion
(AAT1030-1032) in the A-subunit gene and the
(AAAG)n STR alleles in the Thai family. The photograph shows the electrophoretic separation of the (AAAG)n PCR
products from each family member. Repeat alleles have been numbered 1 and 2 exclusively in this family and do not relate to the number of repeats. The symbols for male () and female () are hatched
according to the mutation alleles present.
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To elucidate whether this inframe deletion leads to a loss of catalytic
activity or protein instability, we have introduced the deletion into
an A-subunit cDNA and expressed the recombinant protein in S
cerevisiae. An immunodetectable band was observed in the crude
lysate of yeast expressing the mutant protein (Fig 5); however, this was far weaker than the
level of expression obtained for the normal enzyme in parallel cultures
since the amount of mutant culture lysate applied to the gel was about
four times that of the normal control.
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| Fig 5.
Western blot of normal and deleted A subunits in fresh
lysates of S cerevisiae AH22. Total yeast lysate protein was
prepared from S cerevisiae transformed with lane 1, pGal181/wild-type factor XIII A-subunit cDNA (1.5 µg); lane 2, negative control vector pGal181 (1.5 µg); and lane 3, pGal181/Asn
344-deleted factor XIII A-subunit cDNA (6 µg). The samples were
subjected to SDS-PAGE, blotted onto nitrocellulose membrane, and
developed with antiserum to human factor XIII A subunit.
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|
RNA hybridization analysis (Northern blot) revealed that mRNAs for both
wild-type and mutant factor XIII A subunit were present in equal
abundance in cells grown in galactose but were absent from cells grown
in glucose, consistent with induction from the GAL1 promoter
and no change in mRNA stability caused by the mutation (Fig
6). The transglutaminase activity of the
mutant enzyme determined by 14C putrescine incorporation
into casein was not significantly different to the negative control
of yeast lysate without the recombinant A-subunit plasmid (Table
2). Attempts to purify the mutant protein
via a C-terminal 6xHis tag and immobilized metal affinity
chromatography were not successful. Thus, it was not possible to obtain
a specific activity for the mutant protein. The failure of this
purification appeared to be due to instability of the mutant protein as
normal A subunit can be readily prepared by that procedure. A
pulse-chase experiment was performed to evaluate the stability of the
recombinant protein. After 15 minutes there was significant degradation
of the mutant enzyme and no change in the normal enzyme (Fig
7). Thus, the mutant protein appears to be
both catalytically inactive and unstable.
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| Fig 6.
RNA hybridization analysis of factor XIII A-subunit
expression in yeast. (A) Ethidium bromide stained gel; (B) hybridized membrane. Total RNA was prepared from S cerevisiae AH22
transformed with negative control vector pGal181 (lanes 1, 4),
pGal181/wild-type factor XIII A-subunit cDNA (lanes 2, 5), or
pGal181/Asn344-deleted factor XIII A-subunit cDNA (lanes 3 through 6)
and grown either in YPGal (induced; lanes 1 through 3) or YPD
(repressed; lanes 4 through 6). Hybridization with a factor XIII
A-subunit probe and chemiluminescent detection were performed as
described in Materials and Methods. RNA size standards (Promega) on the
left are in nucleotides.
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Table 2.
Transglutaminase Activity of Fresh Yeast Lysate
Expressing Normal Recombinant Factor XIII A Subunit and the A
Subunit With the Asn344 Deletion
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| Fig 7.
Pulse-chase analysis of recombinant factor XIII A
subunits expressed in yeast. (A) pGal181/wild-type factor XIII A
subunit. (B) pGal181/Asn344-deleted factor XIII A subunit. The cells
were metabolically labeled for 10 minutes followed by a 15-minute
chase.
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The Asn344 deletion occurs within the core domain of the enzyme. Figure
8A shows the relative position of this
deletion with respect to the active center of the A subunit. Comparison
of the wild-type structure to that of the minimized structure shows
good agreement with an overall root mean square (rms) deviation of about 0.7 Å. This measure of structural deviation takes the square root of the average squared Euclidian distance between superimposed atoms from both structures. The rms deviation for the residues from
340-349 show a similar value, and all side chains have similar orientations. In contrast, comparison of the wild-type structure with
that of the minimized deletion mutant shows an rms deviation of about
1.8 Å for residues 340 to 343 and 345 to 349. The largest change
involves the complete reorientation of Asp 343 (Fig 8B), which now
points away from the proposed catalytic triad. This residue is
conserved among transglutaminase sequences that possess the Cys314,
His373, and Asp396 triad. Further, it normally forms a hydrogen bond
with Arg11 in the activation peptide.51 The location of
Asp343, directly in the proposed active site, suggests that its
reorientation as a consequence of the Asn344 deletion would be
detrimental to the enzyme.
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| Fig 8.
(A) Illustration of the active site in the
core domain of factor XIII A subunit. Side chains of active site
residues Cys314, His373, Asp396, and Trp279, Ser340, His342, Asp343,
Asp345, Trp370, Thr398, and Gln400 are shown along with the side chain
of Asn344, which is deleted in the patient studied here. (B) As for Fig
8A but showing a possible configuration for the mutant enzyme. Asp343 has a completely different orientation that is expected to be detrimental for enzyme activity.
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| |
DISCUSSION |
Since the publication of the first reports of mutations in the A
subunit of factor XIII in 1992,21,22 a number of additional mutations have been identified. Among those mutations, six deletions in
the A-subunit gene have been reported (Table
3). Surprisingly, all of them cause
frameshifts resulting in the premature termination of translation. In
this study we have identified the first inframe deletion in the factor
XIII A-subunit gene. The resulting protein has a normal sequence but
lacks Asn344 (which is coded by the missing AAT1030-1032).
The effect of the deletion on the enzyme's expression and activity has
been investigated by in vitro mutagenesis, and its effect on enzyme
structure has been predicted by the computer modeling of the deleted
enzyme.
The result of the Western blot analysis indicates that the deletion of
Asn344 significantly lowers the level of expressed enzyme in yeast
lysates. Because the mutant A subunit mRNA is stable, it is possible to
conclude that the deleted protein might be expressed at a normal level
but is sensitive to degradation. It is notable that although
immunodetectable protein can be observed in lysate of yeast expressing
the mutant enzyme, its activity is not significantly different to that
of the negative control lysate. This implies that the deleted enzyme
definitely loses its transglutaminase activity. The instability of the
mutant protein prevented its successful purification and
characterization in greater detail. These results are in agreement with
data derived directly from the patient. Activity measurement of the
patient's plasma found less than 5% of normal activity. In addition,
a Western blot of the patient's plasma revealed the presence of only a
trace band of A subunit. Because this minimal amount of plasma A
subunit in the patient could be residual normal A subunit remaining
from prior replacement therapy, it appears that the mutant enzyme
prematurely degrades and has a short lifespan in the circulation.
The Asn344 residue is directly adjacent to residues associated with the
active center. The Asn344 deletion occurs on a loop and might otherwise
leave the molecule largely unperturbed but for the fact that other
residues on this loop are directly associated with the active center.
It has been proposed that His342 and Asp343 guide the lysyl substrate
residue into the active site.51 These two residues occur
adjacent, in sequence as well as in the structure, to Asn344 and will
almost certainly be reoriented as a result of a deletion at position
344. Figure 8B shows a possible configuration of these residues that
are also immediately juxtaposed to the proposed catalytic triad:
Cys314, His373, and Asp396. As a consequence, a deletion occurring at
position 344 would be expected to have a profound impact on enzymatic
activity. Such prediction is in agreement with the absence of
transglutaminase activity in the lysate of yeast expressing the deleted
enzyme and the absence of fibrin cross-linking activity in the plasma
of this patient. In addition, this Asn344 deletion could have a serious
effect on the stability of the enzyme.
Because both parents of the patient are heterozygous for the same
AAT1030-1032 deletion, it implies that they may have a
common ancestor despite the apparent absence of consanguinity in this
family. An extensive family study could reveal further information
about the origin of this deletion. As this deletion does not change any
restriction cleavage site, the best method for its identification is
the direct electrophoresis of amplified exon 8 DNA. Under the
conditions described here the 3-bp deletion can be readily resolved. In
addition, further study of this family using the linked polymorphic
short tandem repeat element in the 5 flanking sequence40
revealed the mode of inheritance of this deletion among the immediate
family members (Fig 4). Although the inheritance of the STR allele 1 from the patient's father is equivocal, the deletion allele in the
patient's brother is clearly inherited from the father. Analysis of
the inheritance of the linked STR alleles could be readily applied for
the prenatal diagnosis of the deletion in this family.
Two of the previously reported deletions in the A-subunit gene,
AATT1385-1388 in exon 1131 and AG at the intron
B/exon 3 boundary,22 occurred within repetitive sequence
and might be explained by the slipped strand mispairing mechanism.52 However, the mechanism causing the
AAT1030-1032 deletion in this study and another four
previously identified deletions (a deletion/insertion in exon 3, a 13 bp deletion in exon 3, a 1-bp deletion in exon 2, and the entire
deletion of exon 5)26,30,32,35 are still not clear.
In conclusion we have identified the first inframe deletion in the
factor XIII A-subunit gene. The resulting enzyme has a deletion of
Asn344. This deletion occurs in the catalytic core domain, and computer
modeling suggests that it will have a profound effect on catalytic
activity and may influence stability. The deleted enzyme has been
expressed in yeast and shown to have negligible activity and appears to
be prematurely degraded, thus confirming the structural predictions.
The effects of the deletion identified in this study confirm the
significance of the amino acid residues surrounding the catalytic area
in maintaining the structure, stability, and activity of the enzyme.
| |
FOOTNOTES |
Submitted August 8, 1997;
accepted March 4, 1998.
G.C. is a Postdoctoral Fellow of the Australian Research Council.
R.T.B. holds a QEII Fellowship from the Australian Research Council.
Address reprint requests to Philip G. Board, PhD,
Molecular Genetics Group, John Curtin School of Medical Research,
Australian National University, GPO 334, Canberra, Australian Capital
Territory, 2601, Australia.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract