Xenotransplantation therapies

DNA sequences encoding a porcine Gal.alpha.(1,3) galactosyl transferase and clones containing such sequences are provided. The porcine Gal.alpha.(1,3) galactosyl transferase produces the Gal.alpha.(1,3)Gal epitope on the surfaces of porcine cells. This epitope is recognized by human anti-Gal.alpha.(1,3)Gal antibodies which are responsible for hyperacute rejection of xenotransplanted pig cells, tissues and organs.

This invention relates to xenotransplantation (transplantation across 
species) and is particularly concerned with methods of alleviating 
xenotransplant rejection, maintenance of xenotransplanted tissue in an 
animal, nucleotide sequences useful in xenotransplant therapies, rejection 
resistant transgenic organs, and transgenic animals whose tissues are 
rejection-resistant on xenotransplantation. 
The current shortage of tissues for human transplantation has led to recent 
close examination of xenografts as a possible source of organs. However, 
when tissues from non human-species are grafted to humans, hyperacute 
rejection occurs due to the existence of natural antibodies in human serum 
which react with antigens present in these species, with rejection 
occurring within 10-15 minutes of transplantation. This phenomenon 
depends, in general, on the presence of some or all of antibody, 
complement, neutrophils, platelets and other mediators of inflammation. In 
transplantation of vascularized organs between "discordant" species (those 
in which natural antibodies occur) the first cells to encounter natural 
antibodies are the endothelial cells lining the blood vessels and it is 
likely that activation of these cells is induced by antibody binding to 
xenoantigens or other factors, leading to hyperacute rejection. 
There is considerable uncertainty in the art concerning the nature of 
possible target xenoantigens on xenograft tissues. Platt et al 
(Transplantation 50:817-822, 1990) and Yang et al (Transplant. Proc. 
24:593-594, 1992) have identified a triad of glycoproteins of varying 
molecular weights as the major targets on the surface of pig endothelial 
cells. Other investigators (Holgersson et al, Transplant Proc 24:605-608, 
1992) indicate glycolipids as key xenoantigens. 
We have now found that xenograft rejection, particularly in the context of 
pig tissue, is associated with antibodies reactive with galactose in an 
.alpha.(1,3) linkage with galactose, (the Gal.alpha.(1,3)Gal epitope) 
Modulating the interaction between antibodies reactive with the 
Gal.alpha.(1,3)Gal epitope of xenotransplant tissue effects rejection. 
In accordance with the first aspect of this invention, there is provided a 
method of inhibiting xenotransplant rejection in an animal patient, 
comprising administering to the patient an effective amount of an 
antagonist of antibody binding to xenotransplant antigens having galactose 
in an .alpha.(1,3) linkage with galactose. 
Another aspect of this invention relates to the maintenance of xenograft 
tissue in an animal, which comprises administering to the animal a graft 
rejection effective amount of an antagonist to antibodies which bind to 
the xenograft antigen epitope Gal.alpha.(1,3)Gal. 
In another aspect of this invention there is provided a method of 
inhibiting the binding of antibodies to the Gal.alpha.(1,3)Gal epitope 
which comprises modulating the interaction between the antibodies and the 
epitope with an antagonist which blocks the binding of the antibodies to 
the Gal.alpha.(1,3)Gal epitope. 
Preferably the xenograft recipient is a human. Age is not a determining 
factor for xenograft transplantation although transplants in the elderly 
over 75 years would normally not be carried out. The invention is directed 
particularly to human transplantation with xenograft tissue. 
Xenografted tissue is preferably of pig origin. Tissues from other mammals 
are also contemplated for use in this invention. Preferably the 
xenotransplanted tissue is in the form of an organ, for example, kidney, 
heart, lung or liver. Xenotransplant tissue may also be in the form of 
parts of organs, cell clusters, glands and the like. Examples include 
lenses, pancreatic islet cells, skin and corneal tissue. The nature of the 
xenotransplanted tissue is not of itself critical as any xenotransplanted 
tissue which expresses antigens having Gal.alpha.(1,3)Gal epitopes may be 
utilized in accordance with this invention. 
The binding of antibody to the Gal.alpha.(1,3)Gal epitope expressed on 
xenotransplanted tissue provokes rejection of the tissue by humoral as 
well as cell-mediated immune effects leading to tissue rejection in a very 
short time scale, such as less than one hour. Antagonists which antagonize 
the binding of antibodies to the Gal.alpha.(1,3)Gal epitope block antibody 
binding and therefore inhibit xenotransplant rejection. Because antibody 
binding is blocked, immune responses which give rise to tissue rejection 
are prevented. 
In accordance with a further aspect of this invention, there is provided an 
antagonist which modulates the interaction of antibodies directed against 
Gal.alpha.(1,3)Gal. 
Any antagonist capable of modulating the interaction between antibodies 
directed to the Gal.alpha.(1,3)Gal linkage may be utilized in this 
invention. By reference to modulation, is meant blockage of antibody 
binding or decrease in affinity reactivity of antibodies for the 
Gal.alpha.(1,3)Gal epitope. Various mechanisms may be associated with the 
blockage of antibody binding or decreased affinity of antibodies for their 
respective epitope. These include binding or association with the antibody 
reactive site and change of conformation of the antibody reactive site, 
such as by binding to residues associated with, adjacent to, or distanced 
from the active site, which effect the conformation of the active site 
such that it is incapable of binding the Gal.alpha.(1,3)Gal epitope or 
binds the epitope with reduced affinity. For example, in accordance with 
techniques well known in the art (see, for example, Coligan, et al., eds. 
Current Protocols In Immunology, John Wiley & Sons, New York, 1992; Harlow 
and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 
New York, 1988; and Liddell and Cryer, A Practical Guide To Monoclonal 
Antibodies, John Wiley & Sons, Chichester, West Sussex, England, 1991), 
such a change of the conformation of the antibody reactive site can be 
achieved through the use of an anti-idiotypic antibody raised against the 
natural antibody or fragments thereof. As is also well known in the art, 
these anti-idiotypic antibodies may be modified to enhance their clinical 
usefulness, for example by enzymatic techniques such as preparing Fab' 
fragments, or by recombinant techniques such as preparing chimeric, 
humanized, or single chain antibodies. 
This invention is not limited to any specific antagonist and any antagonist 
which is non-toxic and which modulates the interaction between antibodies 
specific for the Gal.alpha.(1,3)Gal epitope may be used in this invention. 
Suitable examples of antagonists include D-galactose and melibiose, 
stachyose and methyl-.alpha.-D-galactopyranoside, D-galactosamine and 
derivatives thereof. The term derivatives encompasses, for example, any 
alkyl, alkoxy, alkylkoxy, aralkyl amine, hydroxyl, nitro, heterocycle, 
sulphate and/or cycloalkyl substituents whether taken alone or in 
combination, which derivatives have antagonist activities. This may be 
assessed according to methods as herein described. Carbohydrate polymers 
containing one or more of the aforesaid carbohydrate moieties or 
derivatives may also be utilized in this invention. 
The amount of antagonists which is effective to modulate interaction 
between antibodies reactive with Gal.alpha.(1,3)Gal epitopes will vary 
depending upon a number of factors. These include the nature of the animal 
being treated, the nature of species of the transplanted tissue, the 
physical condition of the transplant recipient (age, weight, sex and 
health) and the like. In respect of human transplant recipients of tissue, 
for example from pigs, the amount of antagonists administered will 
generally depend upon the judgement of a consulting physician. As an 
example, a graft rejection effective amount of an antagonist in human 
subjects may be in the order of from 0.01 mg to 1000 gm per dose, more 
preferably 10 mg to 500 mg, more preferably 50 mg to 300 mg, and still 
more preferably 50 mg to 200 mg per dose. 
The schedule of administration of antagonists to inhibit rejection and 
maintain xenografts will depend upon varying factors as mentioned above. 
Varying dosage regimes may be contemplated, such as daily, weekly, monthly 
or the like. 
The mode of administration of antagonists and dosage forms thereof are not 
critical to this invention. Antagonists may be administered parenterally 
(intravenous, intramuscular or intraorgan injection), orally, 
transdermally, or by vaginal or anal routes, or by other routes of 
administration, as are well known in the art. Antagonists may be in solid 
or liquid form and would generally include pharmaceutically acceptable or 
veterinarially acceptable excipients and/or carriers. Examples of dosage 
forms which may be used in this invention are those well known in the art 
as mentioned previously such as described in Remington's Pharmaceutical 
Sciences (Mack Publishing Company, 10th Edition, which is incorporated 
herein by reference). 
In still another aspect of this invention, there is provided nucleotide 
sequences encoding .alpha.(1,3) galactosyl transferase and mutants 
thereof. Preferably, the nucleotide sequence encodes pig .alpha.(1,3) 
galactosyl transferase. 
Nucleotide sequences may be in the form of DNA, RNA or mixtures thereof. 
Nucleotide sequences or isolated nucleic acids may be inserted into 
replicating DNA, RNA or DNA/RNA vectors as are well known in the art, such 
as plasmids, viral vectors, and the like (Sambrook et al, Molecular 
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, 
Second Edition 1989). 
Nucleotide sequences encoding .alpha.(1,3) galactosyl transferase may 
include promoters, enhancers and other regulatory sequences necessary for 
expression, transcription and translation. Vectors encoding such sequences 
may include restriction enzyme sites for the insertion of additional genes 
and/or selection markers, as well as elements necessary for propagation 
and maintenance of vectors within cells. 
Mutants of nucleotide sequences encoding .alpha.(1,3)galactosyl transferase 
are particularly preferred as they may be used in homologous recombination 
techniques as are well known in the art (Capecchi M R, Altering the Genome 
by Homologous Recombination, Science 244:1288-1292, 1989; Merlino G T, 
Transgenic Animals in Biomedical research, FASEB J 5:2996-3001, 1991; 
Cosgrove et al, Mice Lacking MHC Class II Molecules, Cell 66:1051-1066, 
1991; Zijlstra et al, Germ-line Transmission of a disrupted 
B2-microglobulin gene produced by homologous recombination in embryonic 
stem cells, Nature 342:435, 1989) for the inactivation of wild type 
.alpha.(1,3) galactosyl transferase genes. 
Mutant .alpha.(1,3) galactosyl transferase nucleotide sequences include 
nucleotide deletions, insertions, substitutions and additions to wild type 
.alpha.(1,3) galactosyl transferase such that the resultant mutant does 
not encode a functional galactosyl transferase. These nucleotide sequences 
may be utilized in homologous recombination techniques. In such 
techniques, mutant sequences are recombined with wild type genomic 
sequences in stem cells, ova or newly fertilized cells comprising from 1 
to about 500 cells. Nucleotide sequences utilized in homologous 
recombination may be in the form of isolated nucleic acids sequences or in 
the context of vectors. Recombination is a random event and on 
recombination, destruction of the functional gene takes place. 
Transgenic animals produced by homologous recombination and other such 
techniques to destroy wild type gene function are included within this 
invention, as are organs derived therefrom. By way of example, transgenic 
pigs may be produced utilizing homologous recombination techniques to 
produce a transgenic animal having non-functional .alpha.(1-3) galactosyl 
transferase genomic sequences. Tissues derived from such transgenic 
animals may then be utilized in xenotransplantation into human patients 
with the avoidance of immune reaction between circulating human antibodies 
reactive with Gal.alpha.(1-3)Gal epitopes. Such transplants are 
contemplated to be well tolerated by transplant recipients. Whilst 
transplanted tissue may comprise other antigens which provoke immune 
reaction beyond those associated with Gal.alpha.(1-3)Gal epitopes, 
removing the major source of the immune reaction with such transplanted 
tissues should lead to xenotransplants being relatively well tolerated in 
conjunction with standard rejection therapy (treatment with immune 
suppressants such as cyclosporin). 
This invention will now be described with reference to the following 
non-limiting Figures and Examples.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS 
SEQ ID NO:1 Partial nucleotide and predicted amino acid sequence of the pig 
Gal.alpha.(1,3) transferase. 
SEQ ID NO:2 Complete nucleotide and predicted amino acid sequence of the 
pig Gal.alpha.(1,3) transferase. 
SEQ ID NO:3 Nucleotide sequence for PCR primer .alpha.GT-1. 
SEQ ID NO:4 Nucleotide sequence for PCR primer .alpha.GT-2. 
With regard to SEQ ID NOS:1-2, it should be noted that the present 
invention is not limited to the specific sequences shown, but, in addition 
to the mutations discussed above, also includes changes that are found as 
naturally occurring allelic variants of the porcine Gal.alpha.(1,3) 
galactosyl transferase gene, as well as nucleic acid mutations which do 
not change the amino acid sequences set forth in these sequences, e.g., 
third nucleotide changes in degenerate codons. 
EXAMPLE 1 
Materials and Methods 
Cells. Pig cells and tissues were obtained from an abattoir from freshly 
slaughtered animals. Whole blood was centrifuged at 800 g, and 
erythrocytes (RBC) obtained and were washed three times in phosphate 
buffered saline (PBS); pig peripheral blood lymphocytes (PBL) were 
isolated by density gradient centrifugation using ISOPAQUE FICOLL (Vaughan 
et al, (1983) Transplantation 36:446-450). Pig splenocytes were obtained 
from whole spleen by teasing tissue through a sieve to give a single cell 
suspension. Endothelial cell (EC) cultures were established after 
treatment of sterile pig aorta with Collagenase Type 4 (Worthington 
Biochemical Corporation, New Jersey) and the isolated cells were grown in 
Dulbecco's modified Eagles medium (DMEM) (ICN Biomedicals Australasia Pty 
Ltd., Seven Hills, NSW) on gelatin coated plates at 37.degree. C. The 
endothelial origin of EC cultures was verified using rabbit anti human von 
Willebrand factor antibody (Dako A/S, Copenhagen) and indirect 
immunofluorescence. COS cells used were maintained in fully supplemented 
DMEM medium. 
Antibodies. Human serum was obtained from a panel of normal volunteers, 
heat inactivated and pooled before use. The mAb HuLy-m3 (CD48), was used 
as a negative control (Vaughan Supra). Equal volumes of human serum and 5 
to 200 mM 2-mercaptoethanol were incubated at 37.degree. C. for one hour 
to destroy IgM. 
Absorptions. Pooled serum was absorbed with equal volumes of washed, packed 
cells for 15 minutes at 37.degree. C., for 15 minutes at 4.degree. C., 
serum obtained and the procedure repeated three times. For the absorption 
with melibiose-agarose (Sigma, St Louis, Mo.) and sepharose (Pharmacia LKB 
Biotechnology, Uppsala, Sweden), equal volumes packed beads and serum were 
incubated at 37.degree. C. for 16 hours, the beads removed by 
centrifugation, and the absorption repeated several times. 
Serological Assays. a) Hemagglutination: 50 .mu.l of 0.1% pig RBC were 
added to 50 .mu.l of human serum in 96 well plates, incubated at 
37.degree. C. for 30 minutes, room temperature for 30 minutes and on ice 
for 60 minutes prior to both macroscopic and microscopic evaluation of 
hemagglutination; b) Rosetting: Sheep anti human IgG was coupled to sheep 
RBC with chromic chloride and used in a rosetting assay (Parish et al 
(1978) J Immunol. Methods 20:173-183); c) Cytofluorographic analysis was 
performed on FACScan (Becton Dickinson, San Jose, Calif.) (Vaughan et al 
(1991) Immunogenetics 33:113-117); d) Indirect immunofluorescence was 
performed on cell monolayers in 6 well tissue culture plates using 
fluoresceinated sheep anti human IgM or IgG (Silenus Laboratories Pty Ltd, 
Hawthorn, Victoria, Australia) (Vaughan Supra). 
Sugar Inhibitions. Two types of sugar inhibition assays were performed: a) 
50 .mu.l of sugars (300 mM in PBS) were added to 50 .mu.l of doubling 
dilutions of human serum in 96 well plates, incubated overnight at 
46.degree. C. and then 50 .mu.l of 0.1% pig RBC added and the 
hemagglutination assay performed; b) Human serum, diluted in PBS at one 
dilution less than that of the 50% hemagglutination titer, was added to 50 
.mu.l of doubling dilutions of sugars (starting at 300 mM) and incubated 
overnight at 4.degree. C., after which 50 .mu.l of 0.1% pig RBC was added 
and the hemagglutination assay performed. 
Murine Gal .alpha.(1-3) Transferase cDNA construct. A cDNA clone, encoding 
the mouse .alpha.(1,3)galactosyl transferase was produced using the 
published sequence of this transferase (Larsen et al (1989) J Biol. Chem 
264:14290-14297) and the polymerase chain reaction (PCR) technique. 
Briefly two oligonucleotides were synthesized; .alpha.GT-1 (5'-GAATTCAAGC 
TTATGATCAC TATGCTTCAA G-3')SEQ ID NO:3 which is the sense oligonucleotide 
encoding the first six amino acids of the mature .alpha.GT and contains a 
HindIII restriction site, and .alpha.GT-2 (5'-GAATTCCTGC AGTCAGACAT 
TATTCTAAC-3')SEQ ID NO:4 which is the anti-sense oligonucleotide encoding 
the last 5 amino acids of the mature .alpha.(GT and the in phase 
termination codon and contains a PstI restriction site. This 
oligonucleotide pair was used to amplify a 1185 bp fragment from a C57BL/6 
spleen cell CDNA library (Sandrin et al (1992) J Immunol. 194:1636-1641). 
The 1185 bp fragment was purified from a Low Gelling point agarose gel, 
digested with HindIII and PstI (Pharmacia) restriction endonucleases, and 
directionally cloned into HindIII/PstI digested CDM8 vector (Seed B (1987) 
Nature 329:840 842) using T4 ligase (Pharmacia). The product of the 
ligation was used to transform MC1061/p3, and DNA prepared from resultant 
colonies for further examination. One plasmid (p.alpha.GT-3) having the 
1185 bp fragment was selected for further studies. Plasmid DNA was 
prepared, sequenced to confirm the correct DNA sequence, and used for COS 
cells transfection experiments using DEAE/Dextran (Vaughan et al (1991) 
Immunogenetics 33: 113-117; Sandrin et al (1992) J Immunol. 194:1636-1641, 
Seed B (1987) Nature 329:840-842). 
EXAMPLE 2 
Human Anti-pig Antibodies Detect Epitopes Present on Different Cells 
To establish that human serum contains antibodies to pig cells which are 
predominantly of the IgM class, a pool of human serum was made (from 10 
donors) and found to contain antibodies which reacted with pig red cells 
(by hemagglutination); pig lymphocytes (resetting assay and flow 
cytometry); pig spleen cells (resetting); and pig endothelial cells (flow 
cytometry) (FIGS. 1 and 2). Absorption studies demonstrated that the same 
xeno antigens were present on all of these tissues (FIG. 1 and FIG. 2), as 
absorption with RBC, spleen cells or PBL, removed reactivity for the other 
cells (FIG. 1A and FIG. 2). Absorption of the serum pool with EC, while 
removing all of the EC reactive antibodies (FIG. 2a), completely removed 
all PBL reactive antibodies and almost all the RBC hemagglutinating 
antibodies (titer fell from 1/128 to 1/2) (FIG. 1A). Absorption with RBC 
removed 75% (FIG. 2B) and spleen cells all (FIG. 2C) of the EC reactive 
antibodies shown by flow cytometry. Thus, common epitopes are present on 
pig red cells, PBL, spleen and endothelial cells. 
Most of the activity in the serum pool was due to IgM rather than IgG 
antibodies as indicated by the inability of a protein A-sepharose column, 
which does not bind IgM, to alter the titer of the serum after passage 
through the column (FIG. 3), and IgG antibodies eluted from the protein 
A-sepharose column reacted only weakly with RBC (FIG. 3). Furthermore, 
treatment of the serum with 2-mercaptoethanol, which destroys IgM but 
leaves IgG intact, led to a complete loss of antibody activity (FIG. 3). 
When the serum was fractionated by SEPHACRYL gel chromatography, the high 
molecular weight fractions (IgM) were reactive with RBC, whereas the low 
molecular weight fractions (IgG) were not (data not shown). Thus the 
different pig cells carry similar epitopes, all reacted with IgM 
antibodies and in our assays there was little IgG activity found in the 
human serum for pig cells. 
EXAMPLE 3 
Human Anti-pig Antibodies React Predominantly With Terminal Galactose 
Residues 
The ability of different carbohydrates to inhibit the hemagglutination 
reaction (FIG. 4) was examined. Of the sugars tested, inhibition as 
measured by a decrease in titer, was observed with 300 mM galactose, 
methyl-.alpha.-D-galactopyranoside, melibiose and stachyose, all of which 
decreased the titer of the serum pool by 750 (FIG. 4); and with 300 mM 
D-galactosamine, for which a 50% decrease in titer was observed (FIG. 4). 
None of the other monosaccharides tested (listed in the figure legend) had 
any effect on hemagglutination titer (FIG. 4). These studies demonstrated 
that galactose is the part of the epitope, as both melibiose and stachyose 
have terminal galactose residues. It is of interest to note the difference 
in the ability of galactose in the 
.alpha.(methyl-.alpha.-D-galactopyranoside, melibiose and stachyose) but 
not .beta.(methyl-.beta.D-galactopyranoside) configuration to inhibit the 
serum. 
The relative avidity of the antibodies for the sugars which inhibited 
agglutination was estimated from the concentration of sugar giving 50% 
inhibition of the agglutination titer (FIG. 5). Both D-galactose and 
melibiose achieved this inhibition at &lt;1.5 mM, stachyose and 
methyl-.alpha.-D-galactopyranoside at 4.7 mM and D-galactosamine at 18.7 
mM (FIG. 5). By contrast, D-glucose and methyl-.beta.-D-galactopyranoside 
had no effect even at 300 mM concentration. Thus D-galactose is an 
important part of the epitope, as it is a potent inhibitor of the 
xenoantibodies at low concentration (&lt;1.15 mM); the ability of 
methyl-.alpha.-D-galactopyranoside to inhibit agglutination at low 
concentrations (&lt;1.15 mM), compared with the failure of 
methyl-.beta.-D-galactopyranoside (300 mM) to inhibit, demonstrates that 
the galactose residue (which is likely to be a terminal sugar) is in an 
.alpha.-linkage rather than a .beta.-linkage with the subterminal residue. 
The results obtained with melibiose (Gal.alpha.(1,6)Glc) and stachyose 
(Gal.alpha.(1,6)Gal.alpha.(1,6)Glc.beta.(1,2)Fru), which have 
.alpha.-linked terminal galactose residues, are in accord with this 
conclusion. The inhibition of hemagglutination observed with 
galactosamine, which has an additional amine side chain on galactose, (50% 
inhibition of titer at 18.7 mM) could be due to a second carbohydrate 
involved in the epitope, or a lower affinity of the xenoantibodies for 
this sugar. 
To further examine the reaction with galactose, the serum pool was absorbed 
four times with equal volumes of packed melibiose sepharose or with 
sepharose as the control (FIG. 6), one absorption with melibiose-sepharose 
decreased the titer of the antibody from 1/32 to 1/4, and two sequential 
absorptions decreased the titer further to 1/2 (FIG. 6). This absorption 
was specific for melibiose, as using sepharose beads had no effect (FIG. 
6). Thus the majority of the antibody (=94%) reactive with xenoantigens 
reacts with galactose in an .beta.-linkage. 
EXAMPLE 4 
Human Anti-Pig Antibodies React with COS Cells After Transfection with 
.alpha.(1,3) Galactosyl Transferase 
The cDNA coding for the .alpha.(1,3)galactosyl transferase which transfers 
a terminal galactose residue with an .alpha.(1,3) linkage to a subterminal 
galactose has been cloned for both mouse (Larsen et al (1989) J Biol Chem 
264:14290-14297) and ox (Joziasse et al (1989) J Biol Chem 
264:14290-14297). Using this data we used transfection experiments to 
determine the role of the Gal.alpha.(1,3)Gal epitope in isolation of 
others. The mouse transferase was isolated from a cDNA library using the 
PCR technique, and the PCR product was directionally cloned into the CDM8 
vector for expression studies in COS cells. The cDNA insert was sequenced 
in both directions and shown to be identical to the published nucleotide 
sequence (Larsen et al (1989) J Biol Chem 264:14290-14297). COS cells, 
derived from Old World Monkeys, were chosen as they do not react with 
human serum nor with the IB-4 lectin (which is specific for the 
Gal.alpha.(1,3)Gal epitope) (Table 1). After transfection of COS cells 
with the .alpha.(1,3)galactosyl transferase, the Gal.alpha.(1,3)Gal 
epitope was detected on the cell surface by binding of the IB-4 lectin 
(Table 1); these cells were also strongly reactive with the serum pool. 
Absorbing the human sera with pig RBC removed the reactivity for 
Gal.alpha.(1,3)Gal.sup.+ COS cells, (Table 1). Passage of the serum over a 
protein-A sepharose column had no effect on the reactivity of the serum 
for Gal.alpha.(1,3)Gal.sup.+ COS cells, when using an FITC conjugated 
sheep anti-human IgM as the second antibody (this was reflected in the 
same number of reactive cells, the intensity of staining and the titer of 
the serum (Table 1)). In contrast to this, eluted antibodies reacted only 
weakly with the Gal.alpha.(1,3)Gal.sup.+ COS cells, and this reaction was 
only observed when using FITC conjugated sheep anti-human IgG or FITC 
conjugated sheep anti-human Ig, but not FITC conjugated sheep anti human 
IgM (Table 1). Thus human serum has IgM antibodies to the 
Gal.alpha.(1,3)Gal epitope which was expressed on Gal.alpha.(1,3)Gal.sup.+ 
COS cells. The reaction of the serum with Gal.alpha.(1,3)Gal.sup.+ COS 
cells is specific and not due to the transfection procedure as CD48.sup.+ 
COS cells were not reactive with either the serum nor the IB-4 lectin 
(Table 1). Furthermore, the reactivity for both pig RBC (as detected by 
hemagglutination) and EC (as detected by FACS analysis) could be removed 
by absorption with Gal.alpha.(1,3)Gal.sup.+ COS cells but not 
untransfected COS cells. Thus human serum pool contains IgM antibodies 
reactive with the Gal.alpha.(1,3)Gal epitope. 
The level of antibodies in human serum reactive with the Gal.alpha.(1,3)Gal 
epitope can be used to determine the propensity of a patient to 
hyperacutely reject a porcine xenotransplant. In addition, the level of 
such antibodies can be used to determine the amount of antibody antagonist 
that should be administered to a patient prior to such 
xenotransplantation. 
The level of these antibodies can be effectively determined using the 
transfected and untransfected COS cells described above as matched 
Gal.alpha.(1,3)Gal.sup.+ and Gal.alpha.(1,3) Gal.sup.- absorbants, 
followed by a measurement of the reactivity of the absorbed serum for pig 
RBC and/or EC. Higher levels of serum antibody will result in a larger 
difference in reactivity of the serum absorbed against the 
Gal.alpha.(1,3)Gal.sup.+ absorbant versus that absorbed against the 
Gal.alpha.(1,3)Gal.sup.- absorbant. Cells from other species, e.g., human 
cells, can be used in such an assay. Also, rather than using a DNA 
sequence encoding the murine transferase, a DNA sequence encoding the 
porcine transferase (see Example 5) can be used. Such a porcine 
transferase is preferred since there may be differences in the action of 
the murine and porcine transferases, e.g., altered sensitivity to the 
macromolecular environment of the galactose substrate of the enzyme, and 
for a porcine xenotransplantation, it is the level of antibodies against 
the Gal.alpha.(1,3)Gal epitope in the porcine macromolecular environment 
that is of interest. 
In addition to the foregoing, the transfected Gal.alpha.(1,3)Gal.sup.+ 
cells described above can also be used as absorbants to remove 
anti-Gal.alpha.(1,3)Gal antibodies from human serum, e.g., by binding such 
cells to a solid support and passing the serum over the immobilized cells. 
EXAMPLE 5 
Cloning of Porcine .alpha.(1,3) Galactosyl Transferase 
Utilizing the murine cDNA clone for the .alpha.(13) galactosyl transferase 
as a hybridization probe we have cloned the pig .alpha.(1,3) galactosyl 
transferase from a .lambda.GT11 pig spleen cDNA library (Clontech 
Laboratories, Palo Alto, Calif.) according to standard methods as 
described in Sambrook et al (supra). This clone, pPGT-4, has been 
deposited with the AGAL and assigned accession number N94/9030. SEQ ID 
NO:1 shows a partial nucleotide sequence and predicted amino acid sequence 
of pig Gal.alpha.(1,3) transferase as determined by sequencing of clone 
pPGT-4. The sequence shown is incomplete at the 5' end. 
Utilizing the cDNA insert of the pPGT-4 clone as a hybridization probe we 
have also cloned the 5' end of the pig .alpha.(1,3) galactosyl transferase 
from a 5' STRECH pig liver cDNA library in .lambda.gt10, according to 
standard methods as described in Sambrook et al (supra). The insert was 
obtained by the PCR technique using a .lambda. oligonucleotide, and an 
oligonucleotide made to the pig sequence. This PCR product was subcloned 
into SmaI cut pBLUESCRIPT KS.sup.+. This clone, pPGT-2, has been deposited 
with the AGAL and assigned accession number N94/9029. 
SEQ ID NO:2 shows a complete nucleotide sequence and predicted amino acid 
sequence of pig Gal.alpha.(1,3) transferase as determined by sequencing of 
clones pPGT-4 and pPGT-2. The pig transferase has high sequence homology 
with both the murine and bovine .alpha.(1,3) galactosyl transferase genes. 
Both the partial and complete cDNA sequences of SEQ ID NOS:1-2 can be used 
in the xenotransplant therapies discussed above. For example, using 
techniques well known in the art, all or a part of any of the nucleotide 
sequences of SEQ ID NOS:1-2, when inserted into replicating DNA, RNA or 
DNA/RNA vectors, can be used to reduce the expression of the 
Gal.alpha.(1,3) transferase in porcine cells by directing the expression 
of anti-sense RNAs in transgenic cells or animals. See, for example, 
Biotechniques, 6(10):958-976, 1988. 
In addition, as illustrated in the following example, the sequences of SEQ 
ID NOS:1-2 can be used as hybridization probes for the characterization 
and isolation of genomic clones encoding the porcine Gal.alpha.(1,3) 
transferase. Mutants of the genomic nucleotide sequence, in turn, can be 
used in homologous recombination techniques of the types described above 
so that destruction of the functional gene takes place in porcine cells. 
EXAMPLE 6 
Characterization and Isolation of the Porcine Gene Encoding .alpha.(1,3) 
Galactosyl Transferase 
Genomic DNA prepared from pig spleen tissue was digested with EcoR1, BamH1, 
Pst1, HindIII, Kon1 and BstEII, electrophoresed on a 0.8% agarose gel and 
transferred to a nylon filter, the final wash was at 65.degree. C. in 
0.1.times.SSC, 0.1% SDS. As shown in FIG. 7, the genomic Southern blot 
demonstrated a simple pattern suggesting that the gene exists as a single 
copy with a genomic size of .apprxeq.25kb. 
Utilizing the cDNA insert of the pPGT-4 clone as a hybridization probe, we 
have cloned the porcine .alpha.(1,3) galactosyl transferase gene from a 
pig genomic DNA EMBL library (Clontech Laboratories, Inc., Palo Alto, 
Calif.) according to standard methods as described in Sambrook et al 
(supra). This cloning has resulted in the isolation of two lambda phage 
clones, .lambda.PGT-g1 and .lambda.PGT-g5 that contain different regions 
of the porcine transferase gene. 
As discussed above, the gene for the .alpha.(1,3) galactosyl transferase 
can be used to effect targeted destruction of the native gene for this 
enzyme using homologous recombination technology. In accordance with the 
conventional techniques used in this art, such gene knockout is performed 
using fragments obtained from genomic clones of the type provided by this 
example. The gene destruction can be performed in somatic or stem cells 
(Capecchi, 1989, supra). Because such genetically engineered cells do not 
produce the Gal.alpha.(1,3)Gal epitope, they and their progeny are less 
likely to induce hyperacute rejection in humans and are thus suitable for 
xenotransplantation into human patients. 
EXAMPLE 7 
Production of Anti-idiotypic Antibodies Against Human 
Anti-Gal.alpha.(1,3)Gal Antibodies 
Polyclonal anti-idiotypic antibodies against human anti-Gal.alpha.(1,3)Gal 
antibodies are prepared following the procedures of Coligan, et al., 1992, 
supra; Harlow and Lane, 1988, supra; and Liddell and Cryer, 1991, supra. 
Human anti-Gal.alpha.(1,3)Gal antibodies are absorbed from pooled human 
serum onto immobilized melibiose (melibiose-sepharose or 
melibiose-agarose) as described above in Example 3. The antibodies are 
eluted using standard methods, such as, high or low pH, high salt, and/or 
chaotropic agents. Fab' fragments are prepared following dialysis into an 
appropriate buffer. The Fab' fragments are used to immunize rabbits, 
goats, or other suitable animals, along with conventional adjuvants. 
The resulting polyclonal antisera are tested for their ability to change 
the conformation of the human antibody reactive site so as to reduce its 
affinity for the Gal.alpha.(1,3)Gal epitope. Those sera that produce such 
reduced affinity constitute the desired anti-idiotypic antibodies. 
Monoclonal antibodies are produced using the same Fab' fragments as 
antigens to immunize appropriate strains of mice. Hybridomas are prepared 
by fusing spleen cells from such immunized mice with murine myeloma cells. 
Supernatants are tested for antibodies having the ability to change the 
conformation of the human antibody reactive site so as to reduce its 
affinity for the Gal.alpha.(1,3)Gal epitope. Those antibodies that produce 
such reduced affinity constitute the desired monoclonal anti-idiotypic 
antibodies. 
The finding that the majority of xenoreactive IgM is directed to the 
enzymatic product of the single transferase raises the possibility of 
producing transgenic pigs lacking the epitope, by targeted destruction of 
the .alpha.(1,3) galactosyl transferase genes using homologous 
recombination technology. Such genetically modified pigs could be used for 
transplantation. The destruction of the gene is likely to have no 
deleterious effect on the pig--humans live normally in its absence. 
This invention has been described by way of example only and is in no way 
limited by the specific examples herewith. 
DEPOSITS 
Clones pPGT-4, pPGT-2, .lambda.PGT-g1, and .lambda.PGT-g5, discussed above, 
have been deposited with the Australian Government Analytical 
Laboratories, (AGAL), 1 Suakin Street, Pymble, N.S.W. 2073, Australia, and 
have been assigned the designations N94/9030, N94/9029, N94/9027, and 
N94/9028, respectively. These deposits were made under the Budapest Treaty 
on the International Recognition of the Deposit of Micro-organisms for the 
Purposes of Patent Procedure (1977). These deposits were made on Mar. 11, 
1994. 
TABLE 1 
______________________________________ 
Serology On Transfected COS Cells 
Serum Target Reaction.sup.1 
______________________________________ 
NHS GT.sup.+ COS 
+++ 
NHS abs RBC GT.sup.+ COS 
- 
NHS Tx 2-ME GT.sup.+ COS 
- 
NHS abs Protein A 
GT.sup.+ COS 
+++.sup.2 
NHS Eluted Protein A 
GT.sup.+ COS 
+.sup.3 
CD48 GT.sup.+ COS 
- 
NHS CD48.sup.+ COS 
- 
CD48 CD48.sup.+ COS 
+++ 
NHS COS - 
CD48 COS - 
IB4.sup.4 GT.sup.+ COS 
+++ 
IB4 CD48.sup.+ COS 
- 
IB4 COS - 
______________________________________ 
.sup.1 Reactivity detected by indirect immunofluorescence using FITC 
conjugated sheep antihuman Ig or FITC conjugated sheep antimouse Ig unles 
otherwise stated. 
.sup.2 No difference in titer was observed when tested with FITC 
conjugated sheep antihuman IgM. 
.sup.3 Reaction detected on protein A purified immunoglobulin using FITC 
conjugated sheep antihuman Ig or FITC conjugated sheep antihuman IgG, but 
not with FITC conjugated sheep antihuman IgM. 
.sup.4 Reactivity detected using FITC conjugated IB4 lectin. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1353 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Double 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: cDNA to mRNA 
(A) DESCRIPTION: galactosyl transferase, 3' 
clone 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: No 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Sus scrofa 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GTACCGAGCTCGAATTCCGCAAGCCAGTCACCACAAGCCATG42 
ValProSerSerAsnSerAlaSerGlnSerProGlnAlaMet 
505560 
ACTGACCCATGTTCCCCCAGACTGTCGTACCTTAGCAAAGCC84 
ThrAspProCysSerProArgLeuSerTyrLeuSerLysAla 
6570 
ATCCTGACTCTATGTTTTGTCACCAGGAAACCCCCAGAGGTC126 
IleLeuThrLeuCysPheValThrArgLysProProGluVal 
758085 
GTGACCATAACCAGATGGAAGGCTCCAGTGGTATGGGAAGGC168 
ValThrIleThrArgTrpLysAlaProValValTrpGluGly 
9095100 
ACTTACAACAGAGCCGTCTTAGATAATTATTATGCCAAACAG210 
ThrTyrAsnArgAlaValLeuAspAsnTyrTyrAlaLysGln 
105110115 
AAAATTACCGTGGGCTTGACGGTTTTTGCTGTCGGAAGATAC252 
LysIleThrValGlyLeuThrValPheAlaValGlyArgTyr 
120125130 
ATTGAGCATTACTTGGAGGAGTTCTTAATATCTGCAAATACA294 
IleGluHisTyrLeuGluGluPheLeuIleSerAlaAsnThr 
135140 
TACTTCATGGTTGGCCACAAAGTCATCTTTTACATCATGGTG336 
TyrPheMetValGlyHisLysValIlePheTyrIleMetVal 
145150155 
GACGATATCTCCAGGATGCCTTTGATAGAGCTGGGTCCTCTG378 
AspAspIleSerArgMetProLeuIleGluLeuGlyProLeu 
160165170 
CGTTCCTTTAAAGTGTTTGAGATCAAGTCCGAGAAGAGGTGG420 
ArgSerPheLysValPheGluIleLysSerGluLysArgTrp 
175180185 
CAAGACATCAGCATGATGCGCATGAAGACCATCGGGGAGCAC462 
GlnAspIleSerMetMetArgMetLysThrIleGlyGluHis 
190195200 
ATCCTGGCCCACATCCAGCACGAGGTGGACTTCCTCTTCTGC504 
IleLeuAlaHisIleGlnHisGluValAspPheLeuPheCys 
205210 
ATTGACGTGGATCAGGTCTTCCAAAACAACTTTGGGGTGGAG546 
IleAspValAspGlnValPheGlnAsnAsnPheGlyValGlu 
215220225 
ACCCTGGGCCAGTCGGTCGCTCAGCTACAGGCCTGGTGGTAC588 
ThrLeuGlyGlnSerValAlaGlnLeuGlnAlaTrpTrpTyr 
230325240 
AAGGCACATCCTGACGAGTTCACCTACGAGCGGCCGAAGGAG630 
LysAlaHisProAspGluPheThrTyrGluArgProLysGlu 
245250255 
TCCGCAGCCTACATTCCGTTTCGCCAGGGGGATTTTTATTAC672 
SerAlaAlaTyrIleProPheArgGlnGlyAspPheTyrTyr 
260265270 
CACGCAGCCATTTTGGGGGGAACACCCACTCAGGTTCTAAAC714 
HisAlaAlaIleLeuGlyGlyThrProThrGlnValLeuAsn 
275280 
ATCACTCAGGAGTGCTTCAAGGGAATCCTCCAGGACAAGGAA756 
IleThrGlnGluCysPheLysGlyIleLeuGlnAspLysGlu 
285290295 
AATGACATAGAAGCCGAGTGGCATGATGAAAGCGGGCTAAAC798 
AsnAspIleGluAlaGluTrpHisAspGluSerGlyLeuAsn 
300305310 
AAGTATTTCCTTCTCAACAAACCCACTAAAATCTTATCCCCA840 
LysTyrPheLeuLeuAsnLysProThrLysIleLeuSerPro 
315320325 
GAATACTGCTGGGATTATCATATAGGCATGTCTGTGGATATT882 
GluTyrCysTrpAspTyrHisIleGlyMetSerValAspIle 
330335340 
AGGATTGTCAAGGGGGCTTGGCAGAAAAAAGAGTATAATTTG924 
ArgIleValLysGlyAlaTrpGlnLysLysGluTyrAsnLeu 
345350 
GTTAGAAATAACATCTGACTTTAAATTGTGCCAGCAGTTTTCTGA969 
ValArgAsnAsnIle 
355 
ATTTGAAAGAGTATTACTCTGGCTACTTCCTCAGAGAAGTAGCACTTAAT1019 
TTTAACTTTTCAAAAAATACTAACAAAATACCAACACAGTAAGTACATAT1069 
TATTCTTCCTTGCAACTTTGAGCCTTGTCAAATGGGAGAATGACTCTGTA1119 
GTAATCAGATGTAAATTCCCAATGATTTCTTATCTGCGGAATTCCAGCTG1169 
AGCGCCGGTCCTACCATTACCAGTTGGTCTGGTGTCGACGACTCCTGGAG1219 
CCCGTCAGTATCGGCGGAATTCGCGGCCGGGCGCCAATGCATTGGGCCCA1269 
ATTCCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTGTTTTACAA1319 
CCTCGTGACTGGGAAAACCCTGGCCTTACCCAAC1353 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1423 base pairs 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Double 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: cDNA to mRNA 
(A) DESCRIPTION: galactosyl transferase, 
full coding sequence 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: No 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Sus scrofa 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CGGGGGCCATCCCCGAGCGCACCCAGCTTCTGCCGATCAGGAGAAAATA49 
ATGAATGTCAAAGGAAGAGTGGTTCTGTCAATGCTGCTTGTC91 
MetAsnValLysGlyArgValValLeuSerMetLeuLeuVal 
510 
TCAACTGTAATGGTTGTGTTTTGGGAATACATCAACAGAAAC133 
SerThrValMetValValPheTrpGluTyrIleAsnArgAsn 
152025 
CCAGAAGTTGGCAGCAGTGCTCAGAGGGGCTGGTGGTTTCCG175 
ProGluValGlySerSerAlaGlnArgGlyTrpTrpPhePro 
303540 
AGCTGGTTTAACAATGGGACTCACAGTTACCACGAAGAAGAA217 
SerTrpPheAsnAsnGlyThrHisSerTyrHisGluGluGlu 
455055 
GACGCTATAGGCAACGAAAAGGAACAAAGAAAAGAAGACAAC259 
AspAlaIleGlyAsnGluLysGluGlnArgLysGluAspAsn 
606570 
AGAGGAGAGCTTCCGCTAGTGGACTGGTTTAATCCTGAGAAA301 
ArgGlyGluLeuProLeuValAspTrpPheAsnProGluLys 
7580 
CGCCCAGAGGTCGTGACCATAACCAGATGGAAGGCTCCAGTG343 
ArgProGluValValThrIleThrArgTrpLysAlaProVal 
859095 
GTATGGGAAGGCACTTACAACAGAGCCGTCTTAGATAATTAT385 
ValTrpGluGlyThrTyrAsnArgAlaValLeuAspAsnTyr 
100105110 
TATGCCAAACAGAAAATTACCGTGGGCTTGACGGTTTTTGCT427 
TyrAlaLysGlnLysIleThrValGlyLeuThrValPheAla 
115120125 
GTCGGAAGATACATTGAGCATTACTTGGAGGAGTTCTTAATA469 
ValGlyArgTyrIleGluHisTyrLeuGluGluPheLeuIle 
130135140 
TCTGCAAATACATACTTCATGGTTGGCCACAAAGTCATCTTT511 
SerAlaAsnThrTyrPheMetValGlyHisLysValIlePhe 
145150 
TACATCATGGTGGATGATATCTCCAGGATGCCTTTGATAGAG553 
TyrIleMetValAspAspIleSerArgMetProLeuIleGlu 
155160165 
CTGGGTCCTCTGCGTTCCTTTAAAGTGTTTGAGATCAAGTCC595 
LeuGlyProLeuArgSerPheLysValPheGluIleLysSer 
170175180 
GAGAAGAGGTGGCAAGACATCAGCATGATGCGCATGAAGACC637 
GluLysArgTrpGlnAspIleSerMetMetArgMetLysThr 
185190195 
ATCGGGGAGCACATCCTGGCCCACATCCAGCACGAGGTGGAC679 
IleGlyGluHisIleLeuAlaHisIleGlnHisGluValAsp 
200205210 
TTCCTCTTCTGCATTGACGTGGATCAGGTCTTCCAAAACAAC721 
PheLeuPheCysIleAspValAspGlnValPheGlnAsnAsn 
215220 
TTTGGGGTGGAGACCCTGGGCCAGTCGGTGGCTCAGCTACAG763 
PheGlyValGluThrLeuGlyGlnSerValAlaGlnLeuGln 
225230235 
GCCTGGTGGTACAAGGCACATCCTGACGAGTTCACCTACGAG805 
AlaTrpTrpTyrLysAlaHisProAspGluPheThrTyrGlu 
240245250 
AGGCGGAAGGAGTCCGCAGCCTACATTCCGTTTGGCCAGGGG847 
ArgArgLysGluSerAlaAlaTyrIleProPheGlyGlnGly 
255260265 
GATTTTTATTACCACGCAGCCATTTTTGGGGGAACACCCACT889 
AspPheTyrTyrHisAlaAlaIlePheGlyGlyThrProThr 
270275280 
CAGGTTCTAAACATCACTCAGGAGTGCTTCAAGGGAATCCTC931 
GlnValLeuAsnIleThrGlnGluCysPheLysGlyIleLeu 
285290 
CAGGACAAGGAAAATGACATAGAAGCCGAGTGGCATGATGAA973 
GlnAspLysGluAsnAspIleGluAlaGluTrpHisAspGlu 
295300305 
AGCCATCTAAACAAGTATTTCCTTCTCAACAAACCCACTAAA1015 
SerHisLeuAsnLysTyrPheLeuLeuAsnLysProThrLys 
310315320 
ATCTTATCCCCAGAATACTGCTGGGATTATCATATAGGCATG1057 
IleLeuSerProGluTyrCysTrpAspTyrHisIleGlyMet 
325330335 
TCTGTGGATATTAGGATTGTCAAGATAGCTTGGCAGAAAAAA1099 
SerValAspIleArgIleValLysIleAlaTrpGlnLysLys 
340345350 
GAGTATAATTTGGTTAGAAATAACATCTGACTTTAAA1136 
GluTyrAsnLeuValArgAsnAsnIle 
355 
TTGTGCCAGCAGTTTTCTGAATTTGAAAGAGTATTACTCTGGCTACTTCC1186 
TCAGAGAAGTAGCACTTAATTTTAACTTTTAAAAAAATACTAACAAAATA1236 
CCAACACAGTAAGTACATATTATTCTTCCTTGCAACTTTGAGCCTTGTCA1286 
AATGGGAGAATGACTCTGTAGTAATCAGATGTAAATTCCCAATGATTTCT1336 
TATCTGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTC1386 
TGGTGTCGACGACTCCTGGAGCCCGTCAGTATCGGCG1423 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 bases 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Other Nucleic Acid 
(A) DESCRIPTION: PCR primer `GT- 1 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GAATTCAAGCTTATGATCACTATGCTTCAAG31 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 bases 
(B) TYPE: Nucleic Acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Other Nucleic Acid 
(A) DESCRIPTION: PCR primer `GT- 2 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: Yes 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GAATTCCTGCAGTCAGACATTATTCTAAC29 
__________________________________________________________________________