CA2091102C

CA2091102C - Microsomal triglyceride transfer protein

Info

Publication number: CA2091102C
Application number: CA002091102A
Authority: CA
Inventors: John R. Ii Wetterau; Richard E. Gregg; Daru Young Sharp
Original assignee: Individual
Current assignee: Individual
Priority date: 1992-03-06
Filing date: 1993-03-05
Publication date: 2009-05-26
Anticipated expiration: 2013-03-05
Also published as: DE69332041D1; EP0584446A3; ES2178640T3; ZA931601B; DK0584446T4; JP4298801B2; DK0584446T3; ES2178640T5; EP0584446B2; DE69332041T2; HUT67962A; HU218419B; DE69332041T3; EP0584446B1; HU9300627D0; AU3406493A; ATE219514T1; JPH0638761A; CA2091102A1; MX9301269A

Abstract

Nucleic acid sequences, particularly DNA sequences, coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, expression vectors containing the DNA sequences, host cells containing the expression vectors, and methods utilizing these materials. The invention also concerns polypeptide molecules comprising all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, and methods for producing these polypeptide molecules. The invention additionally concerns novel methods for preventing, stabilizing or causing regression of atherosclerosis and therapeutic agents having such activity. The invention additionally concerns novel methods for lowering serum liquid levels and therapeutic agents having such activity.

Description

MICROSOMAL TRIGLYCERIDE
TRANSFER PROTEIN
Field of the Invention This invention relates to microsomal triglyceride transfer protein, genes for the protein, expression vectors comprising the genes, host cells comprising the vectors, methods for producing the protein, methods for detecting inhibitors of the protein, and methods of using the protein and/or its inhibitors.
Background of the Invention The microsomal triglyceride transfer protein (MTP) catalyzes the transport of triglyceride (TG), cholesteryl ester (CE), and phosphatidylcholine (PC) between small unilamellar vesicles (SUV). Wetterau & Zilversmit, Chem. Phys. Lipids a$, 205-22 (1985). When transfer rates are expressed as the percent of the donor lipid transferred per time, MTP expresses a distinct preference for neutral lipid transport (TG and CE), relative to phospholipid transport. The protein from bovine liver has been isolated and characterized. Wetterau & Zilversmit, Chem. Phys.
Lipids a$, 205-22 (1985). Polyacrylamide gel electrophoresis (PAGE) analysis of the purified protein suggests that the transfer protein is a complex of two subunits of apparent molecular weights 58,000 and 88,000, since a single band was present when pudfied MTP was electrophoresed under nondenaturing condition, 2091i.02 DC21 a while two bands of apparent molecular weights 58,000 and 88,000 were identified when electrophoresis was performed in the presence of sodium dodecyl sulfate (SDS). These two polypeptides are hereinafter referred to as 58 kDa and 88 kDa, respectively, or the 58 kDa and the 88 kDa component of MTP, respectively, or the low molecular weight subunit and the high molecular weight subunit of MTP, respectively.
Characterization of the 58,000 molecular weight component of bovine MTP indicates that it is the previously characterized muftifunctional protein, protein disulfide isomerase (PDI).
Wetterau et al., J. Biol. Chem. 2M, 9800-7 (1990). The presence of PDI in the transfer protein is supported by evidence showing that (1) the amino terminal 25 amino acids of the bovine 58,000 kDa component of MTP is identical to that of bovine PDI, and (2) disulfide isomerase activity was expressed by bovine MTP
following the dissociation of the 58 kDa - 88 kDa protein complex.
In addition, antibodies raised against bovine PDI, a protein which by itself has no TG transfer activity, were able to immunoprecipitate bovine TG transfer activity from a solution containing purified bovine MTP.
PDI normally plays a role in the folding and assembly of newly synthesized disulfide bonded proteins within the lumen of the endoplasmic reticulum. Bulleid & Freedman, Naturg =, 649-51 (1988). It catalyzes the proper pairing of cysteine residues into disulfide bonds, thus catalyzing the proper folding of disulfide bonded proteins. In addition, PDI has been reported to be identical to the beta subunit of human prolyl 4-hydroxylase. Koivu gLAL, J. Biol. Chem. M, 6447-9 (1987). The role of PDI in the bovine transfer protein is not clear. It does appear to be an essential component of the transfer protein as dissociation of PDI
from the 88 kDa component of bovine MTP by either low concentrations of a denaturant (guanidine HCI), a chaotropic agent (sodium perchlorate), or a nondenaturing detergent (octyl glucoside) resufts in a loss of transfer activity. Wetterau gL,1L, DC21 a Biochemistrv3Q, 9728-35 (1991). Isolated bovine PDI has no apparent lipid transfer activity, suggesting that either the 88 kDa polypeptide is the transfer protein or that it confers transfer activity to the protein complex.
The tissue and subcellular distribution of MTP activity in rats has been investigated. Wetterau & Zilversmit, Biochem. Sionhvs.
A= $M, 610-7 (1986). Lipid transfer activity was found in liver and intestine. Little or no transfer activity was found in plasma, brain, heart, or kidney. Within the liver, MTP was a soluble protein located within the lumen of the microsomal fraction.
Approximately equal concentrations were found in the smooth and rough microsomes.
Abetalipoproteinemia is an autosomal recessive disease characterized by a virtual absence of plasma lipoproteins which contain apolipoprotein B (apoB). Kane & Havel in The Metabolic Basis of Inherited Disease, Sixth edition, 1139-64 (1989). Plasma TG levels may be as low as a few mg/dL, and they fail to-rise after fat ingestion. Plasma cholesterol levels are often only 20-45 mg/dL. These abnormalities are the result of a genetic defect in the assembly and/or secretion of very low density lipoproteins (VLDL) in the liver and chylomicrons in the intestine. The molecular basis for this defect has not been previously determined. In subjects examined, triglyceride, phospholipid, and cholesterol synthesis appear normal. At autopsy, subjects are free of atherosclerosis. Schaefer gtAL, Clin. Chem..3A, B9-12 (1988).
A link between the apoB gene and abetalipoproteinemia has been excluded in several families. Talmud gIAL, J. Clin. Invest. $2, 1803-6 (1988) and Huang giaL, Am. J. Hum. C`ea net. M, 1141-8 (1990).
Subjects with abetalipoproteinemia are afflicted with numerous maladies. Kane & Havel, supM. Subjects have fat.
malabsorption and TG accumulation in their enterocytes and hepatocytes. Due to the absence of TG-rich plasma lipoproteins, there is a defect in the transport of fat-soluble vitamins such as DC21 a vitamin E. This results in acanthocytosis of erythrocytes, spinocerebellar ataxia with degeneration of the fasciculus cuneatus and gracilis, peripheral neuropathy, degenerative pigmentary retinopathy, and ceroid myopathy. Treatment of abetalipoproteinemic subjects includes. dietary restriction of fat intake and dietary supplementation with vitamins A, E and K.
To date, the physiological role of MTP has riot been demonstrated. la yi=, it catalyzes the transport of lipid molecules between phospholipid membranes. Presumably, it plays a similar role in vivo, and thus plays some role in lipid metabolism.. The subcellular (lumen of the microsomal fraction) and tissue distribution (liver and intestine) of MTP have led to speculation that it plays a role in the assembly of plasma lipoproteins, as these are the sites of plasma lipoprotein assembly. Wetterau & Zilversmit, Biochem. Biop11Y,8,0cta AM, 610-7 (1986). The ability of MTP to catalyze the transport of TG between membranes is consistent with this hypothesis, and suggests that MTP may catalyze the transport of TG from its site of synthesis in the endoplasmic reticulum (ER) membrane to nascent lipoprotein particles within the !umen of the ER.
Olofsson and colleagues have studied lipoprotein assembly in HepG2 cells. Bostrom et=al.,J, Biol. Chem. M, 4434-42 (1988). Their results suggest small precursor lipoproteins become larger with time. This would be consistent with the addition or transfer of lipid molecules to nascent lipoproteins as.they are assembled. MTP may play a rote in this process. In support of this hypothesis, Howell and Palade, J. Ceil Biol. 22, 833-45 (1982), isolated nascent lipoproteins from the hepatic Golgi fraction of rat liver. There was a spectrum of sizes of partioles present with varying lipid and protein compositions. Particles of high density lipoprotein (HDL) density, yet containing apoB, were found.
Higgins and Hutson, J. Lioid Res. 25, 1295-1305 (1984), reported lipoproteins isolated from Golgi were consistently larger than those from the endoplasmic reticulum, again suggesting the 2091102 DC21a assembly of lipoproteins is a progressive event. However, there is no direct evidence in the prior art demonstrating that MTP plays a role in lipid metabolism or the assembly of plasma lipoprotein.

Summary of the Invention The present invention concerns an isolated nucleic acid molecule comprising a nucleic acid sequence coding for all or part of the high molecular weight subunit of MTP and/or intron, 5', or 3' flanking regions thereof. Preferably, the nucleic acid molecule is a DNA (deoxyriibonucleic acid) molecule, and the nucleic acid sequence is a DNA sequence. Further preferred is a nucleic acid having all or part of the nucleotide sequence as shown in SEQ. ID.
NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7and8.
The present invention also concems a nucleic acid molecule having a sequence complementary to the above sequences and/or intron, 5', or 3' flanking regions thereof.
The present invention further concerns expression vectors comprising a DNA sequence coding for all or part of the high molecular weight subunit of MTP.
The present invention additionally concerns prokaryotic or eukaryotic host cells containing an expression vector that comprises a DNA sequence coding for all or part of the high molecular weight subunit of MTP.
The present invention additionally concerns polypeptide molecules comprising all or part of the high, molecular weight subunit of MTP. Preferably, the polypeptide is the high molecular weight subunit of human MTP or the recombinantly produced high molecular weight subunit of bovine MTP.
The present invention also concerns methods for detecting nucleic acid sequences coding for all or part of the high molecular weight subunit of MTP or related nucleic acid sequences.

DC21 a The present invention further concerns methods for detecting an inhibitor of MTP.
The present invention further concerns a novel method for treatment of atherosclerosis, or for lowering the level of serum lipids such as serum cholesterol, TG, PC, or CE in a mammalian species comprising administration of a therapeutically effective amount of an agent that decreases the activity or amount of MTP.
Such agents would also be useful for treatment of diseases associated or affected by serum lipid levels, such as pancreatitis, hyperglycemia, obesity and the like.

Brief Description of the Drawinas Figure 1 shows bovine cDNA clones. The five bovine cDNA
inserts are illustrated. The continuous line at the top of the figure represents the total cDNA sequence isolated. Small, labeled bars above this line map peptide and probe sequences. The open reading frame is indicated by the second line, followed by **
corresponding to 3' noncoding sequences. Clone number and length are indicated to the left of each line representing the corresponding region of the composite sequence. Clones 64 and 76 were isolated with probe 2A, clones 22 and 23 with probe 37A
and clone 2 with probe 19A. Eco Ri linkers added during the cDNA library constniction contribute the Eco RI restriction sites at the 5' and 3' ends of each insert. The internal Eco RI site in inserts 22 and 76 is encoded by the cDNA sequence. The Nhe I
restriction site was utilized in preparing probes for isolation of human cDNA clones (below). The arrows under each insert line indicate individual sequencing reactions.
Figure 2 shows TG transfer activity in normal subjects.
Protein-stimulated transfer of 14C-TG from donor SUV to acceptor SUV was measured in homogenized intestinal biopsies obtained from five normal subjects. The resuits are expressed as.the percentage of donor TG transferred per hour as a function of homogenized intestinal biopsy protein.

Z0911o2 DC21 a Figure 3 shows TG, transfer activity in abetalipoproteinemic subjects. Protein-stimulated transfer of 14C-TG from donor SUV to acceptor SUV was measured in homogenized intestinal biopsies obtained from four abetalipoproteinemic subjects. The resufts are expressed as the percentage of donor TG transferred/hour as a function of homogenized intestinal biopsy protein.
Figure 4 shows TG transfer activity in control subjects.
Protein stimulated transfer of 14C-TG from donor SUV to acceptor SUV in homogenized intestinal biopsies were obtained. from three control subjects, one with chylomicron retention disease (open circles), one with homozygous hypobetalipoproteinemia (solid circles), and one non-fasted (x). The resutts are expressed as the percentage of donor TG transferred/hour as a function of homogenized intestinal biopsy protein. -Figure 5 shows western blot analysis of MTP in normal subjects. An aliquot of purified bovine MTP (lane 1) or the post 103,000 x g proteins following deoxycholate treatment of 23 g of homogenized intestinal biopsies from 3 normal subjects (lanes 2-4) were fractionated by SDS-PAGE and then transferred to nitrocellulose. The blots were probed with anti-88 kDa.
Figure 6 shows western blot analysis of MTP in control subjects. An aliquot of purified bovine MTP (lane 1) or the post 103,000 x g proteins following deoxycholate treatment of 15 g, 25 g, and 25 g homogenized intestinal biopsies from a subject with chylomicron retention disease (lane 2), a subject with homozygous hypobetalipoproteinemia (lane 3), and a non-fasted subject (lane 4), respectively, were fractionated by SDS-PAGE
and then transferred to nitrocellulose. The blots were probed with anti-88 kDa.
Figure 7 shows western blot analysis of MTP in normal subjects with affinity-purified antibodies. An aliquot of purified bovine MTP (lane 1) or the post 103,000 x g proteins following deoxycholate treatment of 34 g (lane 2) or 25 g (lane 3) of homogenized intestinal biopsies from 2 normal subjects were DC21 a fractionated by SDS-PAGE and then transferred to nitrocellulose.
The blots were probed with affinity purified anti-88 kDa.
Figure 8 shows western blot.analysis of MTP in abetalipoproteinemic subjects. An aliquot of purified bovine MTP
(lane 1) or post 103,000 x, g proteins following deoxycholate treatment of 18 g (lane 2), 23 g (lane 3), 23 g (iane 4), 23 g (lane 5) of homogenized intestinal biopsies from four diiferent abetalipoproteinemic subjects were fractionated by SDS-PAGE
and then transferred to nitrocellulose. In lanes 6 and 7, 100 g -of the whole intestinal homogenate (subjects corresponding to lane 4 and 5) was fractionated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with anti-88 kDa.
Figure 9 shows a Southern blot analysis of a gene defect in an abetalipoproteinemic subject. Ten g of genomic DNA from a control, the abetalipoproteinemic subject (proband), and from the subject's mother and father were cut to completion with Taq I, electrophoresed on 1% agarose and transferred to. nitrocellulose.
Southern hybridization was performed using exon 13 cDNA as a probe. Two hybridizing bands in the normal 'lane indicated the presence of a Taq I site in the normal exon 13. One hybridizing band in the abetalipoproteinemic subject lane demonstrated the absence of this restriction sequence in both alleles in exon 13, confirming a homozygous mutation in this subject. The heterozygous state in the mother and father is shown by the three hybridizing bands, corresponding to both the normal and the mutant restriction patterns.
Figure 10 shows= inhibition in MTP-catalyzed transport of TG
from donor SUV to acceptor SUV by compound A described hereinafter. Compound A was dissolved in DMSO and then diluted into 15/40 buffer. Aliquots were added to a lipid transfer assay to bring the compound to the indicated final. concentrations.
DMSO concentration in the assay never exceeded 2 U600 L, a concentration that was independently determined to have minimal effect on the assay. MTP-catalyzed lipid transport was measured DC21 a for 30 minutes at 37 C. TG transfer was calculated and compared to a control assay without inhibitor. Three independent assay conditions were used to demonstrate MTP inhibition by compound A. Assay conditions were: 8 nmol donor PC, 48 nmol acceptor PC, and 75 ng MTP (open circles); 24 nmol donor PC, 144 nmol acceptor PC, and 100 ng MTP (solid circles); 72 nmol donor PC, 432 nmol acceptor PC, and 125 ng MTP (open squares).
Figure 11 shows the dose response of Compound A on ApoB, ApoAl and HSA secretion from HepG2 cells. HepG2 cells were treated with compound A at the indicated doses for 16 hours.
The concentration in the cell culture media of apoB, apoAl and HSA after the incubation period was measured with the appropriate ELISA assay and normalized to total cell protein. The data shown are expressed as a percentage of the control (DMSO
only).
Figure 12 shows the effect of compound A on TG secretion from HepG2 cells. HepG2 celis were treated with Compound A at the indicated doses for 18 hours, the last two hours of which -were in the presence of 5 Ci/mL 3H-glycerol. The concentration of radiolabelled triglycerides in the cell culture media was measured by quantitative extraction, followed by thin layer chromatography analysis and normalization to total cell protein. The data shown are expressed as a percentage of the control (DMSO only), Figure 13 shows inhibition in MTP-catalyzed transport of TG
from donor SUV to acceptor SUV by compound B described hereinafter. Compound B was dissolved in DMSO and then diluted into 15/40 buffer. Aliquots were added to a lipid transfer assay to bring the compound to the indicated final concentrations.
DMSO concentration in the assay never exceeded 2 L/600 L, a concentration that was independently determined to have minimal effect on the assay. MTP-catalyzed lipid transport was measured for 30 minutes at 37 C. TG transfer was.calculated and compared to a control assay without inhibitor. Two independent assay conditions were used to demonstrate MTP inhibition by compound 209110?
DC21 a B. Assay conditions were: 24 nmol donor PC, 144 nmol acceptor PC, and 100 ng MTP (open circles); 72 nmol donor PC, 432 nmol acceptor PC, and 125 ng MTP (solid circles).
Figure 14 shows the dose response of compound B on ApoB, ApoAl and HSA secretion from HepG2 cells. HepG2 cells were treated with compound B at the indicated doses for 16 hours.
The concentration in the cell culture media of apoB, apoAl and HSA after the incubation period was measured with the appropriate ELISA assay and normalized to total cell protein. The data shown are expressed as a percentage of the control (DMSO
only).

Detailed Description of the Invention Definition of terms The following definitions apply to the terms.as used throughout this specification, unless otherwise limited in specific instances.
The term "MTP" refers to a polypeptide or protein complex that (1) if obtained from an organism (e. g., cows, humans, =.), can be isolated from the microsomal fraction of homogenized tissue; and (2) stimulates the transport of triglycerides, cholesterol esters, or phospholipids from synthetic phospholipid vesicles, membranes or lipoproteins to synthetic vesicies, membranes, or lipoproteins and which is distinct from the cholesterol ester transfer protein [Drayna et al=, Nature 3U, 632-634 (1987)] which may have similar catalytic properties. However, the MTP molecules of the present Invention do not necessarily need to be catalytically active. For example, catalytically Inactive MTP or fragments thereof may be useful in raising antibodies to the protein.
The term "modified", when referring to a nucleotide or polypeptide sequence, means a nucleotide or polypeptide sequence which differs from the wild-type sequence found in nature.

20911.02 DC21 a The term "related", when referring to a nucleotide sequence, means a nucleic acid sequence which is able to hybridize to an oligonucleotide probe based on the nucleotide sequence of the high molecular weight subunit of MTP.
The phrase "control regions" refers to nucleotide sequences that regulate expression of MTP or any subunit thereof, including but not limited to any promoter, silencer, enhancer elements, splice sites, transcriptional initiation elements, transcriptiorlal termination elements, polyadenylation signals, translational control elements, translational start site,, translational termination site, and message stability elements. Such control regions may be located in sequences 5' or 3' to the coding region or in introns interrupting the coding region.
The phrase "stabilizing" atherosclerosis as used in the present application refers to slowing down the development of and/or inhibiting the formation of new atherosclerotic lesions.
The phrase "causing the regression of" atherosclerosis as used in the present application refers to reducing and/or eliminating atherosclerotic lesions.
Use and utility The nucleic acids of the present invention can be used in a variety of ways in accordance with the present invention. For example, they can be used as DNA probes to screen other cDNA
and genomic DNA libraries so as to select by hybridization other DNA sequences that code for proteins related to the high.
molecular weight subunit of MTP. In addition, the nucleic acids of the present invention coding for all or part of the high molecular weight subunit of human or bovine MTP can be used as DNA
probes to screen other cDNA and genomic DNA libraries to select by hybridization DNA sequences that code for MTP molecules from other organisms. The nucleic acids may also be used to generate primers to amplify cDNA or genomic DNA using polymerase chain reaction (PCR) techniques. The-DNA

DC21 a sequences of the present invention=can also be used to identify adjacent sequences in the cDNA or genome; for example, those that encode the gene, its flanking sequences and its regulatory elements.
The polypeptides of the present invention are useful in the study of the characteristics of MTP; for example, its structure, mechanism of action, and role in lipid metabolism or lipoprotein particle assembly.
Various other methods of using the nucleic acids., polypeptides, expression vectors and host cells are described in detail below.
In carrying out the methods of the present invention, the agents that decrease the activity or amount of MTP can be administered to various mammalian species, such as monkeys, dogs, cats, rats, humans, UW., in need of such treatment: These agents can be administered systemically, such as orally or parenterally.
The agents that decrease the activity or amount of MTP can be incorporated in a conventional systemic dosage form, such as a tablet, capsule, elixir or injectable formuiation. The above dosage forms will also include the necessary physiologically acceptabie carrier materiai, excipient, fubricant, buffer, antibacterial, bulking agent (such as mannitol), anti-oxidants (ascorbic acid or sodium bisulfite) or the like. Oral dosage forms are preferred, aithough parenteral forms are quite satisfactory as well.
The dose administered must be carefully adjusted according to the age, weight, and condition of the patient; as well as the route of administration, dosage form and regimen, and the desired result. In general, the dosage forms described above may be administered in single or divided doses of one to four times daily.

DC21 a Detailed description of specific embodiments Nucleic acids -The present invention concerns an isolated nucleic acid molecule comprising a nucleic acid sequence coding for all or part of the high molecular weight subunit of MTP. Preferably, the nucleic acid molecule is a DNA molecule and the nucleic acid sequence is a DNA sequence. Further preferred is a nucleic acid sequence having the nucleotide sequence as shown in SEQ. ID.
NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7 and 8 or any part thereof, or a nucleic acid sequence complementary to one of these DNA sequences. In the case of a nucleotide sequence (e.g., a DNA sequence) coding for part of the high molecular weight subunit of MTP, it is preferred that the nucteotide sequence be at least about 15 sequential nucleotides in length, more preferably at least about 20 to 30 sequential nucleotides in length.
The following text shows a bovine cDNA nucleotide sequence (SEQ. ID. NO. 1), a human cDNA sequence (SEQ. ID.
NO. 2), a comparison of the human and bovine cDNA sequences, the bovine amino acid sequence(SEQ. ID. NO. 3), the human amino acid sequence (SEQ. ID. NO. 4), and a comparison of the human and bovine amino. acid sequences. In the sequence comparisons, boxed regions represent perfect identity between the two sequences.

DC21a BOVINE eDNA SEQUENCE
(SEQ. ID. NO. 1) AAAGATGTAA ACC AAAA TGTGAATCAA CP.G.GAC',GAG AGAAGAGCAT 200 CAATGCAAAG ACCTGTGCTC CTTCATCTAA T'I'CATGC'~AAA GATCAAAGAG 300 TTCrACTCAT ATC.AAAATGA ACCAGCAGCC ATAGAAAATC TC'..AAGAGAGG 350 CCTGGCTAGC CTATTTC.AGA TGCAGTTAAG CTCTGGAACT ACCAATGAGG 400 TAGACATCTC TGGAGATTGT AAAG'TGACCT ACCAGC'~C'TC'~ TCAAGAC'.AAA 450 GTGACC.AAAA TTAAGC'~CTTT GGATTCATGC AAAATAGAGA GGGCTGGATT 500 TACGACCCCA CATC'AC'~GTCT TGGG'TGTCAC TPCGAAAGCC ACATCTGTC',A 550 CTACCTATAA GATAGAAGAC AC'~C"r''TG'TrG T'AGC,'T.GI'GCT C'TCAGAAGAG 600 ATACGTGC'IT TAAC?,GC'I'C:4A TTTTC.'TACAA TCAATAGCAG GCAAAATAGT 650 AGCCAGGAAA GCAGGTTGC.A GCCATCATTA AAGCAG'I'CGA TTC'AAAGTAC 750 ACGGCCATTC CCATTGTGGG GCA('3GTC'I'TC CAGAGCAAGT GCAAAGGATG 800 CCCT'I'CTCTC TCAGAGCACT GGC'.AGTCCAT CACAAAACAC C'.LC'~CAGCCTG 850 ACAACCTCTC CAAGGCTGAG GC,"TGTCAGAA GCTTCCTGGC C"TTC.ATCAAG 900 DC21 a Bt?VINE cDNA SEQUENCE

(SEQ. ID. NO. 1, continued) -CACCAGACTC ATTAGACGCC ATTTTC-,GACT TTCTGGATTT CAAAAGCACC 1050 GAGAGCGTTA TCCTCCAGGA AAGGTTTCTC TATGCCTGTG CATTTGCC'I'C 1100 CITT'~GGAAG CAATGACATC AGAGAATCTG TTATGATCAT CATCGGGGCC 1200 CT'I'GTCAGGA AGTTGTGTCA GAACCAAGGC 'I'GC'AAACTGA AAGGAGTAAT 1250 AGAA.GCCAAA kAGT'I'AATCT TGGGAGGACT TGAAAAACCA GAGAAAAAAG 1300 ATGAGGTAAA GAAGACTATG AACAGGATAT ACCACCAGAA TCGT.AAAATA 1500 TCCATCCTAC ATGGAAGTAA AAAACATCCT GCTCTCTATT GGGGAACT'I'C 1600 CCAAAGAAAT GAATAAGTAC ATGCTCTCCA T1'GTCC^AAGA CATCCTACGT 1650 T'I"I`GAAACAC CTGCAAGCAA AATGGTCCGT CAAGTTCTC'~A ACGAAATGGT 1700 CGCTCATAAT TACGAT('GTT TCTCCAAGAG TGGGGTCCTCC TC'1'C',CATATA 1750 DC21 a BOVINE cDNA SEQUENCE
(SEQ. ID. NO. 1, continued) ATTCT'1'TACT CTGGTTCZY`.~G CATTCTAAGG AGAAGTAATC TGAACATCTT 1850 TCAGTATATT GA&AAACTC CTCTTCATGG TATCCAGGTG GTCATTGAAG 1900 AACCTTGACT CCTATGCTGG CTTGTCAGCT CTCCTCTZ"IG ATGTTCAGCT 2000 CAGACCTGTC AC'I7'TTTTCA ACGG~.~'TACAG TGATTTGATG TCCAAAATGC 2050 * TGTCAGCATC TAGTGACCCT ATGAGTG'TG,G TGAAAGGACT TC.'TTC'TGCrA 2100 GTCTATGG'I'A TCGTGAATCT AAAACCCGAG TGAAAAATCG GGTAAGTGTG 2250 GGAAATTGGT GCAGAAACAG AAGCAGGCTT GGAGTTTATC TCCACGG'1'GC 2350 AGTTI"I'CTCA GTACCCATTT TTAGTTTGTC TCCAGATGGA CAAGGAAGAT 2400 GTTCCATACA GGCAG'I'TTC'zA GACAAAATAT GAAAGC'aC'I'GT CCACAGGCAG 2450 AGGTTACATC TCTCGGAAGA GAi4AAGAAAG CCTAATAGGA GGATGTC'~AAT 2500 TCCCGCTGCA CCAAMGAAC TCTGACATGT GCAAGGTGGT GTT'TGCTCCT 2550 TTGC'I'CTCTG AGAGCACAGT GTTTACATAT TTACCTGTAT TTAAGAG'TTT 2700 DC21 a BOVINE cDNA SEQUENCE
(SEQ. ID. NO. 1, continued) TG.rAGAACGT GATGAAAAAC CTCACATAAT TAAGTTTGGG CCTGAATCAT 2750 TT.,ATACTAC CTACAGC''I'C ATTC'1'GAGCC ACTCTATGI'G ATACTTTAGT 2800 AGCGI"1'CTGT TTTCCTGCAT CTCTCTCAAA TCACATTTAC TACTGTGAAA 2850 DC21 a HUMAN cDNA SEQUENCE
(SEQ. ID. NO. 2) ATTCTTCTTG CTG'!'GCTTTT TCTCTGCTTC ATTTCCTCAT ATTCAGCTTC 100 AGCTCACGTA CTCCACTGAA GTTCTTCTTG ATCGC'~GGC'.,AA AGCAAAACTG 200 ATGC'~GGAAT CCTGATGGTG ATGATGACCA GTTGATCCAA ATAACGATGA 300 TGATC.,AAAAT TAAGGCCTTG GATTCATGCA A.AATAGCGAG GTCTGGATTT 650 209110,94 DC21a HUMAN cDNA SEQUENCE
(SEQ. ID. NO. 2, continued) -AATAAGC'7AAG TATTACCTCA GCTGGTGGAT GCTGTCACCT CTGCTCAGAC 1150 CTCAC'~ACTCA TTAGAAGCCA.TTTTGGACTT TTTGGATTTC AAAAGTGACA 1200 CATCCCAATG AAGAACTCCT GAGAGCCCTC ATTAGTAAGT TC'AAAGGTTC 1300 TATTGGTAGC AGTGACATCA GAGAAACTGT TATGA'I'CATC ACTGGC'~ACAC 1350 TI'GTCAGAAA GTTGTGTCAG AATGAAGGCT GC'.AAACTCAA AGCAGTAGTG 1400 GAAGCTAAGA AGTTAATCCT GGGAGGACTT GrAAAAAGCAG AGAAAAAAGA 1450 CACC'T'GGCTA CCACTGCTCT CCAGAGATAT GATCTCCCTT TCATAACTGA 1600 CCAAC`AAATG AATAAATACA TGCTCGCCAT TGTTCAAGAC ATCCTACGTT 1800 T'I'C'-fAAATGCC TCCAAGC'.AAA ATTGTCCGTC GAGTTCTGAA GGAAATGGTC 1850 DC21 a HUMAN cDNA SEQUENCE
(SEQ. ID. NO. 2, continued)-ACCTTGACTC CTATGCTGGT ATGTCAGCCA TCCTCTTTGA TGTTCAC,CTC 2150 AGACCTGTCA CCTTTTTCAA CGGATACAGT GATTTGATGT CC.AAAATGCT 2200 GAGGTCCAGG GTGGTCTAGC TATTGATATT TCAGGTGC'.AA TGGAGTTTAG 2350 GT'1'TTCTCAG TACCCATTCT TAGTTTGCAT GCAGATGGAC AAGGATGAAG 2550 TTGCTCTCTG AGAGC:ACAGC GTTTACATAT TTACCTGTAT TTAAGATTTT 2850 TGTAAAAAGC TACAAAAAAC TGCAGTTTGA TC.AAATTTGG GTATATGCAG 2900 DC21 a NUMAN cDNA SEQUENCE
(SEQ. ID. NO. 2, continued) TAGT'I"ATTCT CTAAGAGGAA ACTAGTGTTT GTTAAAAACA AAAATAAAAA 3050 CAAAACCACA CAAGGAGAAC CCAATTTTGT TTCAACAATT TTTGATt'.AAT 3100 GTATA7.'GAAG CTCTTGATAG GACTTCCTTA AGCATGACGG GAAAACCAAA 3150 CACG'.CTCCCT AATCAGC'~AAA AAAAAAAAAA AAAAA 3185 DC21 a BOVINEMUMAN cDNA SEOUENCE COMPARISON
BOVINE ------------------------------------------ ----------- ----------------HUMAN

BOVINE ------------------------------------------------------------------------HUMAN
GCTTCATTTCCTCATATTCAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTA H

HUMAN :>:.~ ~r::.. =:.:..~~,,..,=:::.,..::.::: ;::> : ,i"~S 225 :~..3`~.'7i~.~: ~...'1.` ~~'=....~~"`ti. ~.~~.^.~. ~.Se.

:. ..:.:..... .. . .:: ~:;. .:
:..::
HUMAN

: ~Ã~.. . ~~~~n. ~~>~t~AC~~ ' t~~~~A`= ~ .
, =~;.,. .
BOVINE ~1 ~'g:<;:.,..x..=;<..:, .,<;<:.,.:Y G ':AT~.=; 226 . .... : :.

.: ::.::>::.:.:>~.,,,::.: ,:.. ~ ,. ,..= :.:.::............
. . :..~.:.. .,.. ...,,....,..:,.....~:~.~`.~..:'~..~.'..~.,........,....
~:o:....,.::ti.:..,:.x=.>.::<~:..:a ..:.;:
BOVINE ~p ~EtTp t' ~. ~ 301 :: ,:=
=.
HUMAN

:~i.,~~,~~,~'.t~,.,~,.>+:.~k... ~;~#A.. ..,r~:: ,.~.... :.?~':<=
=s n:, .,,,........_.,....,..,_ ~~ ti ............. ....: ~:>.:.::.::::.: . .
.:..:;::a:;~:: :5:::::.,:- ':.~::::.:::x.

BOVINE - F..... 376 .,.:=.,:....::.:..,.,..,;:.. z..,~:= :.::::.:.<...::
HUMAN ~1..::.:. ~~A~.:.:.....'.::.`.. .`.~f:A. ..: ~~~.. fa. 525 o-: <..::='~.,.,>.=c~:,'.~.õ=,..,E;~~:,;<o BOVINE ~~~: =: .: ~~~P~~~~.:.::.: ~~>.: ..~~~P~:~1~'~~~~~S~I~A~p~~92.~~~F
.:.::::..::.:. 451 BOVINE <~A$'A~::.:.'. `~ 526 n:r.;t::;:ro=i~::::,:::^.i .::'T...:::,:: =:::::::,:.::....:::.:::: :o:
HUMAN .;,. .:.... : .::.::.: ::;~:.:..~~::.~ 675 -mwGE;AT.::T~~::AC~:::.::

:,.
BOVINE

~x.=e;: ti.,~:::.:.:~::.v. , HUMAN CA~~ =k'<..:.~ ::.~::::: ::: ~ A :~~...~.~:~ . ; .~8 825 ~...,~ ~::~... ~+ ~.:~4~3` ~.~~.. ~~.,.~...~i. .~~=`..
~ r::i:z:;::~ ,.~::z:~ ~~~~~~ ,.. ,.~=,~

~~.,. ~~~...

BOVINE ~~~`~~Y~~'~~~~*'~q~= 826 HUMAN :;.a5:ca. is.zo:: zo:::.:::,..:a~::~.k~ :~.: =.a=~=.'~.::z..`. \ v'::>
s9~'.`~4 975 BOVINE MIX~=<< 901 HUMAN ..........~,.: 1050 ., .
BOVINE .<=;: ~..: C 976 :. =.,=<..=.:: ,;;..
., .: ...::...:,:. .. .:r..
.<:a>...
..~.. .' . ...., :..
.
HUMAN ~ T 1125 :.~.: ,~.~..
.., .... .:. ,~ ..... .::.::a..,~::.

BOVINE : ~-; ~"Y'..:: ~~~~~~. .;: ~~~ .: xTA 1051 : :.:.:.
~.:.:::.:=::::::::.::.., :..
HUMAN ;:.:..; X~~~Cv7~~~ G 1200 :.::~>.:.....:.......~........:...,...<:....:... ... ...
. ......:... .~.::.:. ..~'c......: .:.......... :::::: ...,.. ..... . ;.

BOVINE A ".;TT: 1126 =;: =;::, : . .: : .:.: : : : .
.. ..~. >t..:.:.;=,:,,.,,t~..o.::, BOVINE C:.: IWTM1201 HUMAN ::,::::::::1350 P

209110?
DC21 a BOVINE/}iUMAN cDNA SEOUENCE COMPARISON

9UMAN .Cn`~.'~`...:' 3l~~,;f~?~::::.:};~;..:. ;:~;~i4~:. :;::=3.=;:~:=.:;;.

BOVINE :R:~` ~~F'=3!
: Ts HUMAN :~:; ~A 1351 ~=y:.yy::<;.::.< :.:::..::::::.:: i:...:

BOVINE T :~:.~.~'i`~,'~:.'.;L'~',*.:.A,'.,::..ti,,:~;,,.,:~==.~'.,.~,~,:.
=;=~C~.f'o.,.c,,~l,,C ,:.`t~',,.~v'.`C'~.:L~i.,:','.<,',P4..'~~
~.=i.~..':..4.i .`o~: ..'t~,~P..",~'.;,.,.., 1926 HUMAN E-p G 1575 ,.;~..:.~:=::.,..,,~:.:,,..: ':.}:,C~~~~~;.,..,., ~~...., ::.:.:..
.:.,~.~a.'~:fi ,xtii :}:' FJ Fr::iS'ir.'r'r+J

HUMAN

~~~~=..e=.=...~x,.=.:.,:~x::. ~..:......,.::..,~
,.~~`.>A,.~a.~=+:.`=~=.,=.~i,M~

~=: :=. .:...= =.. ..,..:.~.;: ..~...:, HUMAN ;~i =~ 1725 ,. :.. . :.
BOVINE ;:.:~~P'f* :T~3`~g:;:..: :...~xR~~~~Y~4~::;.. .~;:.:.~"s.;:: <~~$~=.:
~~$C~~A~.`::.::: 1651 :.,`. }:.,;. :.}.,.:,,= :.....:..:.:.,:.,..:,.
HUMAN ,. 1800 -moo .4., BOVINE Y:. Y~3~AiliuCAiSA~Csf~i~~~;:. :. ~9:. G~~1~~.:=:::. 1726 .:.~.:,,:: :tia.r::R~s:..::;:.: .: :::.. . ::~ ~x..:;}::::~:::.::::,~:::=.
:.: c:,:.
: ..
HUMAN

::: < .::::::..::...: ...: :..~}::~;.::.. . ::....,........ ::.. . .
,.....:....:.i ....:..::.~ ::::... .:: :... ............. . ,:.::, ..

s: ;::::=s~~~~: :.:> >:;:: .;=:;;:..,;
.. ..
... ..

:::. ::;::: = .:}:::~ =:}, . .......:...
..s.: :;:: .:;::< ..: ........::.,.::. .:::=,:.:::::::.~. :::.::<.: ,>.:~:..:.

:=.~i: :=, ... .
~;.;<>',> :;:::'#`:~~....~:~~=1l4~.:..::. 2025 :.. :
HUMAN
.: .:... :..:.: .. =
= :.:.:.,~:::y.,:..:..~::::. ::,:::::...
....:... ::: ::.:::..: , .

BOVINE :.~-~5:.:.:.:: '~~}~~~'a~'~'~`r~':r~a3::<:.: ~?~,. ,:::-..;~~~^.:;,:;`.

~. = :}:::::;<>=: : ::i:;;<
==
HUMAN ;, : .: =. = :......}.. '..: , ..4.:v.y::i:(w:.:~'.::i:: : :v. :... ~u .n .:vn. , =hY

BOVINE d~~}~~~~4~~":: ~~::<i.:...:: ~~~~~~~~~~~~?~~~-: ~.<,:..:
~.>:.::.;.'::.:.,.. 2026 ..>a.. . ~.,. .:::.:}.
... =:....:....: .::.:
. . . .
:.. . . :. .
HUMAN

= .......c...,,,.:=,..::,,.,.:.~==:,::.}:..,..,:..:. ~ :..: ::. ' ::a:::., ..........:.. ::.::.:.::<:::, ..,....:,. :..:,::...... .. .z __..

BOVINE ~~j~fi~j~~.,:=:= ~ry~ 2101 :.s:::}:::=:;::<:,<:i :.::::;:.:::: :.. , ~~~'~::' ~~~ : . ~~~~~~~ ~ ~~ : `
HUMAN :xz a::E~:t' 2250 ..~.~,~.. ,.~,.. ..~ : ..;~~.. .~=õ'~.,i:.

HUMAN o:~f~=e. :i:~ ~~ ::: ~~xwT::
: .::.::.:...:: :5~::,::,. :~~~:~.~M.. ~r ~4t~~~- ~:.:.:: 2325 BOVINE lZ:~E"n~S~;~= ~ . 2251 : :::k: <::.=Y.<.: :.. :.::.}:.=.'.}::..::.. .: <>:...:...
: :.. , i..:.~,=.

::}: ~:.. =.:,,..:,. .y..,,,.....,...... ..,..,:. :,.,,, BOVINE 6 ~1~q=..'.:.: T 2326 .s~~ '= 'J :}=.f>% : }õi,~~~ Cti'1.'!.:A' >iift. '.:{:/ '!h::::.'{l:G .~i .4'i...,=..:). .h.. ., nhvtt?"=!y.' :..=

::.. ......:.... ,.e:ti=z. ..,o ..ti:. .:.~., BOVINE ,. ~~`;~'~`9"~~~G~"'r.:=.:': P:.` fi'xL~A~'..: 2401 .::=}:..: .,...~+: :....::...::::....
HUMAN :u~~~~'`t `~~:'. '. :~:~~~:~~: .~.=.~,"`~. =..~'~.~'..~""...E.'.''A~.:
:_~,,,.:':~.:,:..>..~ :}:~..:~~=:.~~,:.:~'..~.A.``..
A~ 2550 ..~`...1=~#.. :.~.. ...,~,~,.. ..,~=i=.=:,~.; ~~.y..:..,:~ .,~:,=.

BOVINE õS~=;==, ~~3~x~~G~3l~~~~~~, L~~S; 2476 ::.: ......
a.
HUMAN

..~4: . .:~:= ;.=:::..~~ t.~~.....~...A~~:~~a`3FG......... ~. ..
Nxlm BOVINE T.2$~,';:. 2551 ..;~.=:.}=>r:: ,.::.::::: ::y:.
, ., .:.
HUMAN :.... :};

2001102 DC21a BOVINE/HUMAN cDNA SEQUENCE COMPARISON

BOVINE G ~ 2626 HUMAN C
~~ . ~~~...~~..... -T. 277 ~~

: ~=:.:::..a:.;
HUMAN ^:.::~=,:~.:.=.~=~=.:~.=:::;::=>.:=.;:,::.:::.:.;. .::.~...:. :::.~::.~
i;:..c.;::.: 2850 .........:

BOVINE C A~ ;:~;,...; ~no 2774 HUMAN T 3)i 2922 .~.
~ Ms B

OVINE ~~ ~ R
..::<..:.: .:;
HUMAN :5`:,~',.: ~ .,. ;. ~;:~~. 2995 ~;

BOVINE qfflmmmlw- -------------------- 2900 HUMAN T:,, a>:.::, CAAAACCACACAAGGAGAAC 3070 BOVINE ---------------------------------------------------------------- -------HUMAN
CCAATTTTGTTTCAACAATTTTTGATCAATGTATATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAA

DC21 a BOVINE PROTEIN SEQUENCE
(SEQ. ID. NO. 3) KDVNLLENVNQ QRGEKSIFKG KKSSQIIRKE TTLEAYQRPVL LHLIHGK'CFZE 100 FYSYQNEP.AA Ia1LIqRa1S LFQMQLSSGT TNE.~'VDISGDC KVTYQAHQDK 150 VTKIKALDSC KIERAGFfi1.P HQVLGV'TSKA TSVTTYKIID SFWAVLSEE 200 IRALRLNFT.Q SIAQiIVSRQ KLFL,RTTEAS VRLKPG~QVA AIIKAVDSKY 250 TAIPIVGQVF QSKCKGCPSL SEHWQSIRKH LQPDNLySKAE AVRSFLAFIK 300 HLRTAKKF.,EI I-QILEAENKE VLPQLVDAVT SAQTPDSLDA ILDFLDFKST 350 ESVILQERFL XACAFASHPD EET T RAT.TSK FKGSFGSMI RESVMIIIGA 400 LVFtKLjCQNQG CKKGVIEAK KLILGGIM EKKEDIVMYL LALRIA~E 450 GIPT r,T x= TCEGPISFiLA. ATTLQRYDVP FI'II)EVKCIM NRIYHCMKI 500 HEKI'VRTTAA AIILENNPSY MEVKNILLSI CEIPKEMKY MLSIVQDILR 550 FPASKMt7R QVVIKEMVAHN YDRFSKSGSS SAYTG'YVERT SHSASTYSLD 600 NLDSYAGLSA LSFDVQL,RPV TFFNGYSDLM S104LSASSDP MS'WKGLLLZ, 700 IDHSSn,ELQLQ SGL,K'ANMC)VQ GGLAIDITGA MEESLWYRES ICTRVlWVSV 750 LITGGITVDS SFVM=IG AETEAGLEFI STVQFSQYPF LUCLUVK 800 VPYRQFETKY EEtLSTGRGYI SRKFUMSLIG GCEFPLHQEN SDM':'KWFAP 850 DC21 a HUMAN PROTEIN SEQUENCE
(SEQ. ID. NO. 4) MILIAVLFL,C FISSYSASVK GHTTGLSLNN DRLYKLTYST EV=RGT~GK 50 IQDSVGYRIS SNVDVALLWR NPDGDDDQLI QITMVNVE NVNQQRC{.S 100 IFKGKSPSKI NtatFntr FAr Q RPTr ,r =uLIHG KV=SYQN EAVAIIIVM 150 GLA5LEQTQL SSGT'INEVDI SGNCKV'1'YQA HQDKVIKIKA LDSCKIARSG 200 FTTPNQVL,GV SSKATSVT'i'Y KIE:DSFVIAV LAEETHNFGL NELQTIKGKI 250 VSKQKLELKT TEAGPRLM,SG KQAAAIIKAV DSKYTAIPIV GQVFQSHCKG 300 FNKE77PQLV DAVTSAQI'SD SLEAILDFLD FKSDSSIILQ ERFILYACC~'A 400 SHPrEEr,TRA LISKEZiGSIG SSDIRETVNE ITGTLVRK,C QNF",GQ=V 450 VEAI4{LILGG TEKAEKKEDT R&rLLAJIM LLPEGIPSLL KYAEAGF= 500 SHr ATTA? QR YDLPFITDEV KKMRIYHQ M2KVf-IEKTVR TAAAAIII,NN 550 NPSYMVRIVI LLSIGELPQE NM;YMLAIVQ DILRIZ',PAS KIVRRVIKE24 600 FQYIGIQ~GLH GSQVVIFAQG LEALIAATPD EC,'EENn nSYA CVJSAILFDVQ 700 LRPVTFFNGY SDIMSK@ff,SA SGDPISWKG LILLIDHSQE LQLQSGIKP,N 750 IEVQGG.IAID ISCAMEFSLW YRESKTRVKN RV''I'WITTDI TVDSSFVnG 800 RGYVSQKRKE SVLAGCEFPL HQENSEMQ{V VFAPQPDSTS TGWF 894 DC21 a HUMAN PROTEIN SEQUENCE
(SEQ. ID. NO. 4, continued) MILLAVLFI,C FISSYSASVK GHTTGLSLNN DRLYKLTYST EVLLDRGKGK 50 LQDSVGYRIS SNVDVALLWR D?PDGDDDQLI QITMEIDVNVE NVNQQR=S 100 IFKGKSPSKI . RPTLLHLIHG KVKEF'YSYQN EAVAIEIVIKR 150 GZASLFQTQL SSGZ"I'NE'VDI SGNC=QA HQDKVIKIKA LDSCKIARSG 200 VSKQKLELRT TEAGPRuh.SG KQAAAIIKAV DSKYTAIPIV GQVFQSHCKG 300 CPSLSELWRS TpjZYIQpDNL SKAEAVRNFL AFIQHLRTAK KEEILQILKM 350 ENF=QLV DAVTSAQTSD SLEAILDFLD FKSDSSIILQ ERFLYACGFA 400 SHPTMT,TRA LISKFKGSIG SSDIRETVMI ITGTLVR= QNEGCKLKAV 450 VF.,P,KKLILGG LEKAII~'..DT R=AMa LLPEGIPSLL KYAEAGEGPI 500 SHi,A'TTATQR YDLPFITDEV HIKTLNkIYHQ NRF~IVR T TILNN 550 NPSYNIDVMI LLSIGELPQE MKYMLAIVQ DILRFEMPAS KIVRRVI= 600 VP,HNYDRFSR SGSSSAYTGY IERSPRSAST YSLDILYSGS GILRRSNI.NI 650 FQYIGKAGLH GSQVVII~'~QG LEALIAATPD EGEQNLDSYA G'ISAILFDVQ 700 LRPV'=NGY SDIMSIdMLSA SGDPISWKG LILLIDHSQE IQLQSG= 750 IEVQ C,GLAID ISC'zlEFSLW YRESKTRVKN RVTWITTDI TVDSSFVKAG 800 LETSTETEAG LEFISTVQFS QYPFLVa4j-I DKDEAPFRQF EKYERLSTG 850 RGYVSQ~= SVIACC=L HQENSEM~V VFAPQPDSTS SGfiF 894 DC21 a BOVINE/HUMAN PROTEIN COMPARISON

BOVINE ---------- ---------- ---------- --- ..".:..... .... 16 HUMAN MILLAVLFLC FISSYSASVK GHTTGLSLNN DRL =, s~il~~':` .;.,, ~if~~~,. 50 :.. .. . . _.. . . _...

:~:.A~~~: . ~::;;~..: :~i"~~ :' :~2~~:;=.:. ?~~?Q~ =
<:Y :<.:
HUMAN ~ `.+'~t~~: ~lx :s 10 0 ;:::':ca.y:=yyy~.+yYy+:=o-:.xoY::.x=.yy:.:y: . ,.~.~=õs., ,~>\~~.=~,.~~~~.~=,~.~,~,~'=~.,.,,,'~.bt:,..
::[=. . ...\ :..............t........
:...... .:...:v: .............:.

?>\:\GVx4' =.\:`.'::`v\\\ .'Y,.)h~ `=f\Y:\.. `:\\"t:~ ~ . \~:.,~`~\.
='ti`:~'.=. =.\'.3 :4\f \' \\= eY:= \\\ \` ,.,, :~EY)v.2.:`.t'Y~v\..= ~..\ \. :.... : . ~~:yYr\-'\: t ~:C:R
;

k~~:
.l~?~`~~:.'.;
h+ \~ \\ \h,k...:~~+ ~==v*\ACV1\v.:::,v:Y.},.\~\ v~v,.\ \::,\~..\E,\:,.yYVyP .
bi\:uni~Y,\.6t:~\~,:\:.4.vu HUMAN +~,~.+ ~Y ?~U"i~'~S~'~~~~v.. '=~~ : v .' : :: ::=:: ;L~~'' 200 2'ttC:N:E.Y:;A>\u :t:=.\`y;a't:t=`.yv.yY:pS:o:::Y:>.::..Sqv :4..,:.::Y
,.::t::Y:v+Y.a::.:YYT.:
.~~'u', }~1~.ib :............ .::: ..`; ..
BOVINE ~~`` RAL 216 .`.tt v. <::~::' HUMAN .::~`; 5~ Y '~~..:'~ =`~~:.. ~: HNF 250 . ..v.
:.:.:: =.=.. : : .' y :'::::<. .,.,== ,..:. ...:. .:~;.>=::.: :.:::. .:.. ........:.>:
.... .... ;;:...
BOVINE ~t~ :' !]! ;'~' <<='~` ~'~K~~;`D~~~~'~3~FZ~7<sii':"..r > ~ 266 >y~ H "'=t~""~ti~#~.'~'3~~=~~.<:~y :...:.:..:..... ~.~s:~
%i=Yff'`

.?~~..:.Y. . ..~?~::..'.~~~..
y:iy:y ... ............ .. ....: ............
BOVINE :~+...F~ 316 :,:=:Y::: ~; .:... .
:?::k:r'<:: r::{;: ..r:..:ayY.=>y.`;:=:,uYY::::;:t, :~.s.:s.,ax::::::~
.vt:t:~:Y.4yy~o,Y:."'\"th\t:;,':.
f 3 f `~ t ~i:i~: ':;>..:C..<r.::i: %;::}:a:=7yy:.k4:i '.:4::i,~vti :::::::t\ y ::t..o:na.:q~y>
..................: ..........+,. ..;... ,.:,:1~: . ..~y....
BOVINE X~IiF= 'F3:~~" +A~';~?`'="''~ ~=+,~ ~`~' `...:~:~`::: ~_~~ .'~ 366 S?~i Y'P = ~. '..a:;;., ~ . `:~=.:>;= ~ ,:.~' ~~i....: .:; .
':::i:x: <..xu<.::::Y=`:s,: ~Y;`y::i:.:: E::v.x<::.:,::::: :.
a:..iL.yY::....u\:::+'::<::y;:ti. v :~~ ~ i:' Q'.u~y.y.Y;'::~ : \v .,=.:;`.:::o ay:\y:: :.::vY:\.:;::,.: ..:~ iu5:o BOVINE 'w4t.. 416 ::~::~`Y
:<.:::
HUMAN .: :.~k;i~i~$~~' 450 :..:u},.Y: :nkAt=`, :~:.\. YY:\.\`.`~~t+:`. \\\~F::\=\u\\\~

BOVINE
% 500 HUMAN
4.r.=:..<u.YY::Y~¾~a~}~~~t~~. ::~ :i\\:iV\4.> t~~`a T+t~.:M.; ~ ~~~v~
..=.\\~h=:.`+.:;

BOVINE p'.~ .>:` ~`~' `~F~ i ' ~a:.+ :~`~'~~ = . ~.
.==4':i?i'.f~.~i :. G`\l` ..
};i}::<{L::; .. ..,~}v.i :4.yk?t=y:i:v.iy :v:=::'v:rn \:~~.{r.,s~ u~''v'vC=:\:
.:\ ti . : y . . ., ., :. .

. ...... .. = y. :.
::::=:<.y:<:=>. . :.:<.u.:Yy:YY:.Y::.Y:.. :.Y::: . ....::::.::..:,<Yw~ :;..
..: :..:::<'.Y:.: ::Y:.Y':+ ::.:.>::<:.Y:v.:..::
:........... . : .................. :.........v.,:::::.: ~ ......
:.....,:.:::.: .
..:.........:....
.
BOVINE .

.~~::',=='~z~ssti;.;.y..;~~,: :y..y; :Yk;\u ~:~ `:::.
..YY +<.:w=..,:.:: .:~..~.~,i.:~ ~s=~+ ~ .a..,v HUMAN ;.;~~~: 600 <f'.' ::.a.......... ~;..;

.... :..... ................ .......... ... . .. ...
S~;'~:?~.
BOVINE Y.~~ S~~a~~~+:S~s~ .:a(`~8'`= 616 õ~:.;Y=. :..4..,=::.u.. .:. ::=:
: :~ Y.~:.Y:y:=::.:y ~':.: :....=: =: ...+.
yy:y;:=:;:.::.~::yy.::.,:.y'.:'~y..~~?. =::+v,::< i;
:, ~.:.: ~.. .
...:: ..:...........:...:.. ,:.,.:::::;,:::y:..;::==::...;.:~::.;;., y:=:::.:
............,.:.,...,:.,.....:..
PS~`~ 650 HUMAN
~'i""""r......:'ri:r:::;:'.::::1 ~y:=yy:'~~=::=-:`,=.'~:=r:=:~=:=:=:~='.='.=~:.`:.:~=:='.'='.:rr~'.:.~u".='='.:~y".
n..k;cyy:=yy:oyY:oy:.,:.y::?.yy:yyy::Y::o.::`.:::.y::<:`::.Y::.y:}:.,:y.~:::.YY
:Y:,,y:.

2091102 DC21a BOVINE/HUMAN PROTEIN COMPARISON
_:... ..... _.._ _::...

;~~:: >~t~~`:..=
=;=~;.::
::...~?.:::..i.: =<:;>:s: ~:z<:: =~<>;:::: >.::~ <: :>;:::>? _::<~
::<:~>::;:>:;<.:;a?;::...:

H
;~:SC=. .>~.~t~::
~

DI L It 5~a <
BOVINE ::.~'~ . >~s;=i>::..:t7< ' .`:~ .' . S>~E:;::.õ~:; : >::<i;:=::
..........
::.... ;.:::?:.:
.,:=:?:a>.a<:?::?:..:-:?~:~:<zz;;~ii:~~:>:.::..:.:.? :...:?.....?.:....:.:,.
:.:?~ .::
HuMP,N Q:~`::...:.. 750 ::~==:

, =
.;:

:.~~:;<~ .' ~~:',',.~`. ~;....=.:,.; .:~:.?. `:...i:~:.::.:;.~.?:??:::.,,.
~>.'~ .::::;:::::.?:.;::
HUMAN I ~:s='a t~~'~~ .:~;a ~~$~ ~. ~'.. ~r?~Q~~tS~~ 800 :i::\;:\ i2\\;?>u'?:.
2:?\<:p~::?'fi':=:k`:.i\`:u\ti????:h:t.yi~::<i=`.<ti:<.:v'}: ::t?:=:

.........: .... .........
...: .. . . .......: ............ .. 4 :..

;7S 2<Fi: ~
~' , =:.:='.'~.:~.:
BOVINE G ::~.::: ::..~.~'.`~~.~~z;;S~';.=,..~'~,.:,'.::;.:. ,~;~
.:. ,.~~.?=z: ~::.: >.,:<,:: :::,.:<;
D~
S a~~l~<=:~;~s~5'~:.:: ~'i~N`~~~`~i.' Ep.

~ ~~ ::<::~:~::~>:>' ==;>'<' E~r~ t~:::.._~~""""`~~~~~'"~''' ##.^=~~ `= ~~:'S~`t<#~i.?~?'.:~:: ~ . ~a ss~~~::: ?: ::<;<::<:::>: s:a:; ?:=?::::,:
:`~

DC21 a The bovine cDNA is a 2900 base composite of the cDNA
sequences of clones 2 and 22 and has an open reading frame between bases 1 and 2580, predicting a translation product of 860 amino acids, followed by a TGA stop codon, 298 bases of 3 prime non-coding sequence, and a poly A region.
In the human cDNA, the 3185 bases predict an 894 amino acid translation product from bases 48 to 2729, followed by a TGA
stop codon, 435 bases of 3 prime non-coding sequence, and a poly A region.
In the cDNA comparison, there is about an 88% identity between overlapping sequences in the coding region (bovine bases 1-2583 and human bases 150 - 2732). It is not necessary to introduce any gaps to attain this alignment within the coding region. The homology is somewhat weaker in the 3' noncoding region, including the introduction of several gaps to obtain optimal alignment.
The bovine protein sequence (SEQ. ID. NO. 3) is the 860 amino acid translation product of the combined sequence of bovine cDNA clones 2 and 22. Sequences for the peptide fragments used to design oligonucleotide probes are as follows:
peptide 19A is found between residues 37 and 51, peptide 37A
between residues 539 and 550, and peptide 2A between residues 565 and 572.
The human protein sequence (SEQ. ID. NO. 4) is the 894 amino acid translation product of human cDNA clone 693.
In the amino acid comparison, the bovine protein shows about 86% identity to the human translation product. When considering highly conserved substitutions at nonidentical residues, the two proteins are about 94 % homologous.
The inventors extended their knowledge of the 5' end of the foregoing bovine cDNA sequence with the sequence shown below, 5' to 3'. The top line shows the nucleotide sequence (SEQ.
ID. NO. 5), and the bottom line the amino acid sequence (SEQ. ID.
NO. 6). The new sequence obtained (83 bases) is underlined.

DC21a mm TTT CTC TGC TTC ATT TCC TCA TAT TCA GCT TCT
F L C F I S S Y S A S
GTT AAA GGT CAC ACA ACT GGT CTC TCA TTA AAT AAT
V K G H T T G L S L N N
GAC CGA CTA TAC AAA CTC ACA TAC TCC ACT GAA GTT
D R L Y K L T Y S T E V
The inventors have also extended their knowledge of the 5' end of the foregoing human cDNA sequence. The additional sequence (SEQ. ID. NO. 7) is as follows:
AGAGTCCACTTCTCA
This sequence extends the 5' end of the human MTP cDNA
sequence by 15 bases. These sequences were generated from human liver cDNA clone 754 isolated during the initial human cDNA cloning (see Example 3), but were characterized after clone 693.
The inventors have also elucidated a partial human genomic DNA sequence (SEQ. ID. NO. 8) for the high molecular weight subunit of MTP as shown below. Vertical lines indicate intron/exon boundaries. Exon sequences are in plain type, intron sequences in bold. Arrows indicate portions of the introns for which the sequence is not reported (arrow lengths do not indicate the size of the introns). The numbers in the right column indicate the first and last base of each exon relative to the human cDNA
sequence shown supra. This extended genomic nucleic acid, as well as the extended cDNA, and fragments thereof are useful in the present invention.

-32- DC21a (SEQ. ID. NO. 8) AAGGTTCCTGAGCCCCACTGTGGTAGAGAGATGCACTGATGGTGAGACAC
CATGTTCCCTTACAATGAAAP,CTGGATATGTGTCATTATCTTTAT~ i09 TCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGIIIT

GTGGGCTACCGCATTTCCTCCAACGTGGATGTGGCCTTACTATGGAGGAA
TCCTGATGGTGATGATGACCAGTTGATCCAAAT.AACG~GGGC.ATTTTCT 296 ACCAGATAAATGCAAAGATTAGATATCAGAAGTTTTTGGAGAAGTGTACC
ATTGGACAGCACTTGTATTGGGTTCCCGTTTATAATCCATTAGTTTCTTA
TCTATCACT TCTTTGTTTTAAGGTTTGGTGATGAAAG ----TTATTTTAAGCCTAAAGTCACAGAGTTCTTTAAGTATTGCTATTTTTGCC
TTATTAAAAAACCTAGTTTATAAATACCTTCTCCATTCTTTTAAAGTGAG
TGGCAACGTCCTATAAATCATGAATTGABAAAT TTGTGG
CCAACTCTTTCTGTTTCTTTATCATTTTATTTTCAGAGATACTCTGATGA
AGACAGATATAGGAAGTTTTTTTTAACAGCTTTCTTTCTGTTACTCCA+297 TGAAGGATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGC

ATCTTCAAAGGAAAAAGCCCATCTAAAATAATGGGAAAGGAAAACTTGGA

AGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAA+TAAAGG 440 GGCGTTTAGATTCCACAACTTTTTCTCCAACTTCATATTTTTCTTCCCTT
CAGTAGATATTATTTTGAGGTAATCACATTGTAACTACTTTTATGGTAAA
TGGAATTTCTTCAAGAACTAAAGAiACAGAGGTTGTAAATTAAATGTTTCC
AAACTGAATCAATGCCCTGAGTTCCCTTACATTTACTAGCCAATTTGTTT
CCTATTTTTCTGGAAATCTTTATAGTGGAATGAAGTATTTATTTATTGAT
GAAAGGC.ATTATTAAAAGGTAAATTTCTCATCAAATTATAAGGGATTACA
AACATAATGT TCATCAAAGC'ATGATTGGATGAATTC

-33- DC21a (SEQ. ID. NO. 8, continued) TCTGATAAATGATGCATTTTTGCTTCATTTGTGTTCTGTTCCCCTCTCCC
CACCAC~GTCAAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGA 441 GAACCACCAATGA+TACTTACCAATATTAATAAGGATTCAGCATCTCAA 548 CTAAGGTAATGCTCAGAAAAGGTGACTTGTGTAG
TCCCCTATGGCCTATTAGAGACCTCAATTTTCAAGCCACTTCTCACTAGA
ATTCAAATGGCCCACAAGGEIATCCCAAGCATTATGCCCTTGCCTTTCTTT
TTA+TAGATATCTCTGGAAATTGTAAAGTGACCTACCAGGCTCATCAAG 549 GGATTTACGACCCCAAATCAG~TATGATAGATGTCACTTTCTTTGAGGCA 665 TTAAAATAATTACATTTTGTAC,AGACTAATTTA 10 CGATGATTACTTGTTATAAAGATGGCTATTTATTTATTTAfTCTTGGGT 666 GTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCTT

TGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCC

TACAAACCATTAAGGGGAAAATAGTATCGA+TAAGATAATGCTAAAFITT 805 TTTATTTTCTTTGCTATTCTTTGTTATATTATTATACTTGATTTGT -01' ATGATTATAATATAGCATTTCCCTTTGGTATTATGCA+CAGAAATTAGA 806 GCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAGGCTG

CAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTG

CAAATATGGGAATAATCATGACATCAGACTCTGTTTTCATTTTGTCTCCA
GTGAAAGCATCAACTCATTCA

-34- DC21a (SEQ. ID. NO. 8, continued) GGAGAACACCCTTTGTAAATGTGGATGTTCACAGTTATGAGTGGGGTATG
AGCCTGCAGTGTATGTTTTGCA*TCTCGGAGCTCTGGCGGTCCACCAGG 957 AAATACCTGCAGCCTGACAACCTTTCCAAGGCTGAGGCTGTCAGAAACTT

=CCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAAGAGATCCTTC

AAATACTAAAGATGGAAAATAAGGAAGTATT~TAAGTTCCCCAACCTTTG 1114 TG1'GGGGTTGTCTGTCAGAAACATTTCTGGAGTG
GATATCCATGATTATGCCTTTTTTTATA*CCTCAGCTGGTGGATGCTGT 1115 CACCTCTGCTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGG

ATTTCAAAAGTGACAGCAGCATTATCCTCCAGGAGAGGTTTCTCTATGCC

TGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT~T 1283 AAGTC~TAGAAAATAAAGACCCTCAACTCCTATAAAACTTCTT IAAGAA
TATTAACAGTAATTAAAAGTTTCTTAGATCCGAATTCTTCGCCCTATAGT

CTATTTTATCCCTGGGTGGTTAATA+GTAAGTTCAAAGGTTCTATTGGT 1284 AAAGTTGTGTCAGAATGAAGGCTGCAAACTCAA+TAAGTGCAAATCCAA 1391 TCTCATGTATTACATCATTCTACACCATTGTCCATTTGATACTCACCATG
CTGCCTACTATTGGCACTCCTAATTCTCT7,'TACTCTATTCTACTTACCTT
ATTTGNATAGCAAT

-35- DC21a (SEQ. ID. NO. 8, continued) AACACAATATGCCCATTATTGATAATACTCATTGCTTCTTAAGAATGTAT

TGCTTTTATTCCACTGTGTGCCTCAGTCAAGCAACCAATGCAAAACTTTG
TAAAACTGTAGGTTGCTTTCTTGGACCCAAGAATAAA['~CCAGTCTCACCC

C~GCAGTAGTGGAAGCTAAGAAGTTAATCCTGGGAGGACTTGAAAAAGC 1392 AGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCC

TGCTTCCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAA

GGGCCCATCAGCCACCTGGCTACCACTGCTCTCCAGAGATATGATCTCCC

I TGCTGAACATGAGTCE1PiATGCAPiATTCCGCTCA -AGTCACTCTGTATTTTCCCCAAATAGTCTTCTCTCCTGCTTAAAAATAAC
TCTTAAATTGCATTTGGGGCTATTCTAA
ATGTTTAATTTCTCAGGCTATGCCTAATGTGCATAAGGAAGTATGTGGTC
TGAAGTTCACTACAGTCAT TGGAGAAAGCCACCAGCTC
TTAACGGCCTCAGCCTAGAAGTGATCCTCATAGATTCTATCCATGGCGTA
TTAGCCPiGAACTAGTCACGTGGCCCCCACCAAATCACAAAGGAATCTGGG
AAATGTAGTAACACATGTATATTTTTATGAACACTCACTATTCCTGCTAT
TCCTGCTGAAATGTCCATTTTAAAAATCTAGATGTGCACTAAGTTTGAAC
ATCTTATGAACAC~TGAAGAAGACCTTAAACAGAATATACCACCAAAACC 1605 GTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTA

GGAGCTTCCCCAAGAAATGAATAP.ATACATGCTCGCCATTGTTCAAGACA
TCCTACGTTTTGAAATGCCTGCAA+TATAATACATTGCACATGTCTCTC 1816 TGTGTATTCAAGCTTATTTGTGTGTTCATGGGGTACCGATGTAGCTAATA
ATAATGATGTGGTCATTATGCAA

-36- DC21a (SEQ. ID. NO. 8, continued) AGCTGGACACCCTTGCCTTGCTGTCATTTTGATAGCAAACTAAATTTCAA
ATATCTGAGTAloiTGAA('~GGGCTAGCCCTAATCCTGATGCTACCACGCCAG
CTGGCACCACCCTGGCTCTTGGAAAGGCATGAGGAAAATTTGGCTTCCTC
TTTTTTCCACTGAGGATTTTTTTTTTCCAIATTTGACTTGGGAAACP,GTC
ATTACAATGAATGTGCAGCTTTTTTTTTCCTCATATGTTGCA+AAAATT 1817 CAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGAAC~TATGTACA 1914 C TCTCCTTCCATACCCCACAACTTAGCATTGCTGGAACT
GCTATTAAATTACAGTTATTGTGTGTCATCA+TAGTCCCCGTTCGGCAT 1915 CTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGA

AGTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAG

CCAG~TAACTCACTTCTCATGGATTTTGCTTAATAAAGTATGCAAGAAAT 2036 CAGGCITGAGGTAAAATAAAACATATATGCTGTGGGTAATGCTATAGAATG
TATAAGTTAATGGTGGCTTCTGTCATATTTTGCCCATGATTTCCTTATCT
GTAAGAGGCTGTATGGTTTATAGTCACTCAGAGAAAGTTTCGAATTTGAA
CTTGAAACCTAAGTAATTTGATCCATTGAACTTGACAAATGTCCATT--TGGCCCCTTGAGAAGTTCTAGCTGCAGCTCAGAAGCTTCACCATTATTTA
CAGAGCAGGCAGGGAGCTTGCGTCATGAACATTATATTGATTTTATCCAG

CGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTATGTCAGCCATCCTCT

ATGTCCAAAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGG
ACTTATTCTGCTAATAGATCATTCTCACi3TAATTCANYCAGTCTGTGAGT 2264 ATTTATTGAGTCCCTAAACTACGCCAGGCACGTA -_37_ DC21a (SEQ. ID. NO. 8, continued) ATCAACACAACTCAAATGGAATTATCTACAGCAGGAGGTCAAATGTNCCA
TTGGAAAGGGGGTTAACTAAATTGTACTTATTATTTTTATAACTATTATT
ATGCTTTTTTCTTCTA+AACTTCAGTTACAATCTGGACTAAAAGCCAAT 2265 TAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAG~TAAGTGT 2389 CTTGGGTATTTCTGACCTGCTGAGAGGACCTGGGTTCCAAGAATGTTTTT
CATTTTGGTCTTTGTTATGCCCATACGAAACAATGTAGTATCTTACAGAC
ACTCCCCACATCTGCAACTGAAGGCAC'~GGGAGAGCT
ACCTTCCCTGCCCAATATCTGAGACTCACCAGGCCCTGGTTACCAGCAGA
ACTCTAAGC'ACATCCAGGTCACCTCTGAATCCCTTAAGTGTTTCCTTCCA
GTCACTGGCATCATACGTTCAGACCCTGTAAAGTTACA('~CTGTTAGTCCA
ATACCATTAAATATAATATGAACAAGTTTTTTCTTTTTTTCTCAAATGTT
TAtGTGACTGTGGTAATAACCACTGACATCACAGTGGACTCCTCTTTTG 2390 TGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAGCAGGTTTGGAGTTT
EXON, 17 ATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGAT

GGACAAGGATGAAGCTCCATTCA+TAAGATGCAGCGTACAGGTCATGTT 2560 CCAGGACCATCCCCAGTGCACCAGGAACTTGCATTCAGTTTAGAACATTC
AGTTTCAGBATTAAAACAAAACAGTAGAAACCCAGGGAAAGATGAATTTT
CTTTAAATGAGTAGAAGAATAATTGATAAGGCCAAA}1}1AAGTCAGTTTCT
GGGATACC1hAAAAAAAATCTAATGACTAGTTCATGTGATTCTGGAGATAG
TTATCATATTCTAATCCAGAAACAATTT

-38- DC21a (SEQ. ID. NO. 8, continued) TGCTTTGGAACAGAAACTTCAAGTACATTCAGTAACTTGGCTGGAC',AGGT
ATAGGGTGACTTAACTGTGTGTGTAATTCTGTTAATGTTGCTGTTGTTGT
ACA+CAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTAT 256 1 GTCTCTCAGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCT
CCATCAAGAGAACTCAGAGATGTGCAAAGTGGTGTTTGCCCCTCAGCCGG
ATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTACTTG
AATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTC

TCTGAGAGCACAGCGTTTACATATTTACCTGTATTTAAGATTTTTGTAAA

AAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGTATATGCAGTATGCT
ACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTT
AGTTTACTTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTA
TTCTCTAAGAGGAAACTAGTGTTTGTTAAAAACAAAAATAAAAACAAAAC
CACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGTATAT
GAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTT
CCCTAATCAGG GGTAGGACACAACCAACCCAT
TTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGGAGAAGGGACAAGACT
TTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTT
ATTGTTAGTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATCTCA
TCTTTCTGCTGACCATTTTAGTGAGGTTGTTCCAAAGACATTCAGGTCTC
TACCTCCAGCCCTGCAAAAATATTGGACCTAGCACAGAGGAATCAGGAAA
ATTAATTTCAGAAACTCCATTTGATTTTTCTTTTGCTGTGTCTTTTTGAG
ACTGTAATATGGTACACTGTCCTCTAAGGGACATCCTCATTTTATCTCAC

-39- DC21a (SEQ. ID. NO. 8, continued) CTTTTTGGGGGTGAGAGCTCTAGTTCATTTAACTGTACTCTGCACAATAG
CTAGGATGACTAAGAGAACATTGCTTCAAGAAACTGGTGGATTTGGATTT
CCAAAATATGAAATAAGGAAAAAAATGTTTTTATTTGTATGAATTAAAAG
ATCCATGTTGAACATTTGCAAATATTTATTAATAAACAGATGTGGTGATA
AACCCAAAACAAATGACAGGTCCTTATTTTCCACTAAACACAGACACATG
AAATGAAAGTTTAGCTAGCCCACTATTTGTTGTAAATTGAAAACGAAGTG
TGATAAAATAAATATGTAGAAATCATATTGAATTC

2091102 DC21a The nucleic acids of the present invention can be isolated from a variety of sources, although the presently preferred sequences have been isolated from human and bovine cDNA and human genomic libraries. The exact amino acid sequence of the polypeptide molecule produced will vary with the initial DNA
sequence.
The nucleic acids of the present invention can be obtained using various methods well-known to those of ordinary skill in the art. At least three alternative principal methods may be employed:
(1) the isolation of a double-stranded DNA sequence from genomic DNA or complementary DNA (cDNA) which contains the sequence;
(2) the chemical synthesis of.the DNA sequence; and (3) the synthesis of the DNA sequence by polymerase chain reaction (PCR).
In the first method, a genomic or cDNA library can be screened in order to identify a DNA sequence coding for all or part of the high molecular weight subunit of MTP. For example, bovine or human cDNA libraries can be screened in order to identify a DNA sequence coding for all or part of MTP. Various cDNA
libraries, for example, a bovine small intestine lambda gt10 library (Clontech Laboratories, Inc. Palo Alto, CA), a human liver lambda UNI-Z.APTM XR library (Stratagene Cloning Systems, La Jolla, CA), or a human intestine lambda gtl 0 library (Clontech), can be used.
Various techniques can be used to- screen genomic DNA or cDNA libraries for target sequences that code for the high molecular weight subunit of MTP. This technique may, for example, employ a labeled single-stranded DNA probe with a sequence complementary to a sequence that codes for the high molecular weight subunit of MTP. For example, DNA/DNA
hybridization procedures may be used to identify the sequence in the cloned copies of genomic DNA or cDNA which have been denatured to a single-stranded form. Suitable probes include cDNA for the high molecular weight subunit of MTP acquired from DC21 a the same or a related species, synthetic oligonucleotides, and the like.
-A genomic DNA or cDNA library can also be screened for a genomic DNA or cDNA coding for all or part of the high molecular weight subunit of MTP using immunoblotting techniques.
In one typical screening rnethod suitable for the hybridization techniques, a genomic DNA or cDNA library is first spread out on agarose plates, and then the clones are transferred to fitter membranes, for example, nitroceiiuiose membranes. The genomic library is usually contained in a vector such as EMBL 3 or EMBL 4 or derivatives thereof (e.g., lambda DASHTM). The cDNA
library is usually contained in a vector such as ;Lgt10, %gt11, or lambda ZAP. A DNA probe can then be hybridized to the clones to identify those clones containing the genomic DNA or cDNA coding for all or part of the high molecular weight subunit of MTP.
ARernativeiy, appropriate L gl strains containing vectors %gt11 or lambda ZAP can be induced to synthesize fusion proteins containing fragments of proteins corresponding to the cDNA insert in the vector. The fusion proteins may be transferred to filter membranes, for example, nitrocellulose. An antibody may then be bound to the fusion protein to identify all or part of the high molecular weight subunit of MTP.
In the second method, the nucleic acids of the present invention coding for all or part of MTP can be chemically synthesized. Shorter oligonucleotides, such as 15 to 50 nucleotides, may be directly synthesized. For longer oligonucleotides, the DNA sequence coding for the high molecular weight subunit of MTP can be synthesized as a series of 50-100 base oiigonucieotides that can then be sequentially ligated (via appropriate terminal restriction sites) so as to form the correct linear sequence of nucleotides.
In the third method, the nucleic acids of the present invention coding for all or part of the high molecular weight subunit of MTP can be synthesized using PCR. Briefly, pairs of synthetic 2091102 DC21a DNA oligonucleotides generally at least 15 bases in length (PCR
primers) that hybridize to opposite strands of the target DNA
sequence are used to enzymatically amplify the intervening region of DNA on the target sequence. Repeated cycles of heat denaturation of the template, annealing of the primers and extension of the 3'-termini of the annealed primers with a DNA
polymerase results in amplification of the segment defined by the PCR primers. Bel White, T.J. gtaL, ?rends Genet. 5,185-9 (1989).
The nucleic acids of the present invention coding for all or part of MTP can also be modified (i.e., mutated) to prepare various mutations. Such mutations may change the amino acid sequence encoded by the mutated codon, or they may be silent and not change the amino acid sequence. These modified nucleic acids may be prepared, for example, by mutating the nucleic acid coding for the high molecular weight subunit of MTP so that the mutation resufts in the deletion, substitution, insertion, inversion or-addition of one or more amino acids in the encoded polypeptide using various methods known in the art. For example, the methods of site-directed mutagenesis described in Taylor, J. W. et al., Nucl.
Acids Res. JI, 8749-64 (1985) and Kunkel, J. A., proc. Natl. Acad.
ScL USA 22, 482-92 (1985) may be employed. In addition, kits for site-directed mutagenesis may be purchased from commercial vendors. For example, a kit for performing site-directed mutagenesis may be purchased from Amersham Corp. (Arlington Heights, IL). In addition, disruption, deletion and truncation methods as described in Sayers, J. R. gLaL, Nucl. Acids Res. lfi, 791-800 (1988) may also be employed. Mutations may be advantageous in producing or using the polypeptides of the present Invention. For example, these mutations may modify the function of the protein (e.g., resutt in higher or lower activity), permit higher levels of protein production or easier purification of the protein, or provide additional restriction endonuclease recognition sites in the nucleic acid. All such modified nucleic DC21 a acids and polypeptide molecules are included within the scope of the present invention. ~
Exoression vectors The present invention further concerns expression vectors comprising a DNA sequence coding for all or part of the high molecular weight subunit of MTP or a protein complex comprising both the high and low molecular weight subunits or portions thereof. The expression vectors preferably contain all or part of the DNA sequence having the nucleotide sequence shown in SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7 and 8. Further preferred are expression vectors comprising one or more regulatory DNA sequences operatively linked to the DNA sequence coding for all or part of the high molecular weight subunit of MTP. As used in this context, the term "operatively linked" means that the regulatory DNA sequences are capable of directing the replication and/or the expression of the DNA sequence coding for all or part of the high molecular weight subunit of MTP.
Expression vectors of utility in the present invention are often in the form of "plasmids", which refer to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto. The expression vectors of the present invention may also be used to stably integrate the DNA sequence encoding the high molecular weight subunit of MTP into the chromosome of an appropriate host cell (e.g.,COS or HepG2 cells).
Expression vectors useful in the present invention typically contain an origin of replication, a promoter located 5' to (i.e., upstream of) the DNA sequence, followed by the DNA sequence coding for all or part of the high molecular weight subunit of MTP, DC21 a transcription termination sequences, and the remaining vector.
The expression vectors may also include other DNA sequences known in the art, for example, stability leader sequences which provide for stability of the expression product, secretory leader sequences which provide for secretion of the expression product, sequences which allow expression of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium), marking sequences which are capable of providing phenotypic selection in transformed host cells, sequences which provide sites for cleavage by restriction endonucleases, and sequences which allow expression in various types of hosts, including but not limited to prokaryotes, yeasts, fungi, plants and higher eukaryotes. The characteristics of the actual expression vector used must be compatible with the host cell which is to be employed. For example, when expressing DNA
sequences in a mammalian cell system, the expression vector should contain promoters isolated from the genome of mammalian cells, (e.g., mouse metallothionien promoter), or from viruses that grow in these cells (e.g., vaccinia virus 7.5 K promoter). An expression vector as contemplated by the present invention is at least capable of directing the replication, and preferably the expression, of the nucleic acids of the present invention. Suitable origins of replication include, for example, the Col El, the SV40 viral and the M13 orgins of replication. Suitable promoters include, for example, the cytomegalovirus promoter, the lac Z
promoter, the gal 10 promoter and the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter. Suitable termination sequences include, for example, the bovine growth hormone, SV40, lac Z and AcMNPV polyhedral polyadenylation signals. Examples of selectable markers include neomycin, ampicillin, and hygromycin resistance and the like. All of these materials are known in the art and are commercially available.

DC21 a Suitable commercially available expression vectors into which the DNA sequences of the present invention may be inserted include the mammalian expression vectors pcDNA! or pcDNA/Neo, the baculovirus expression vector pBlueBac, the 5 prokaryotic expression vector pcDNAII and the yeast expression vector pYes2, all of which may be obtained from lnvitrogen Corp., San Diego, CA.
Suitable expression vectors containing the desired coding and control sequences may be constructed using standard 10 recombinant DNA techniques known in the art, many of which are described in Sambrook, gLa., Molecular Cloning: A Laboratorv Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Habor, NY (1989).

15 Host cells The present invention additionally concems host cells containing an expression vector which comprises a DNA-sequence coding for all or part of the high molecular weight subunit of MTP. See, for example the host cells of Example 4 20 hereinbelow, which are preferred. The host cells preferably contain an expression vector which comprises all or part of the DNA sequence having the nucleotide sequence substantially as shown in SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, and the first 108 25 bases of 2 together with 7 and 8. See, for example, the expression vector appearing in Example 4 hereinbelow, which is preferred. Further preferred are host cells containing an expression vector comprising one or more regulatory DNA
sequences capable of directing the replication and/or the 30 expression of and operatively linked to a DNA sequence coding for all or part of the high molecular weight subunit of MTP.
Suitable host cells include both prokaryotic and eukaryotic cells.
Suitable prokaryotic host cells include, for example, E. coli strains HB10i, DHSa, XL1 Blue, Y1090 and JM101. Suitable eukaryotic DC21 a host cells include, for example, Soodo tt~ fruqiperda insect cells, COS-7 cells, human skin fibroblasts, and Saccharomvices cerevisiae cells.
Expression vectors may be introduced into host cells by various methods known in the art. For example, transfection of host cells with expression vectors can be carried out by the calcium phosphate precipitation method. However, other methods for introducing expression vectors into host cells, for example, electroporation, liposomal fusion, nuclear injection, and viral or phage infection can also be employed.
Once an expression vector has been introduced into an appropriate host cell, the host cell may be cultured under conditions permitting expression of large amounts of the desired polypeptide, in this case a polypeptide molecule comprising all or part of the high molecular weight subunit of MTP.
Host cells containing an expression vector that contains a DNA sequence coding for all or part of the high molecular weight subunit of MTP may be identified by one or more of the following six general approaches: (a) DNA-DNA hybridization; (b) the presence or absence of marker gene functions; (c) assessing the level of transcription as measured by the production of mRNA
transcripts encoding the high molecular weight subunit of MTP in the host cell; (d) detection of the gene product immunologically;
(e) enzyme assay; and (f) PCR.
In the first approach, the presence of a DNA sequence coding for all or part of the high molecular weight subunit of MTP
can be detected by DNA-DNA or RNA-DNA hybridization using probes complementary to the DNA sequence.
In the second approach, the recombinant expression vector host system can be identified and selected based upon the presence or absence of certain marker gene functions (e.g., thymidine kinase activity, resistance to antibiotics, etc.). A marker gene can be placed in the same plasmid as the DNA sequence coding for all or part of the high molecular weight subunit of MTP

209110?
DC21 a under the regulation of the same or a different promoter used to regulate the MTP coding sequence. Expression of the-marker gene indicates expression of the DNA sequence coding for all or part of the high molecular weight subunit of MTP.
In the third approach, the production of mRNA transcripts encoding the high molecular weight subunit of MTP can be assessed by hybridization assays. For example, polyadenylated RNA can be isolated and analyzed -by Northern blotting or a nuclease protection assay using a probe complementary to the RNA sequence. Afternatively, the total RNA of the host cell may be extracted and assayed for hybridization to such probes.
In the fourth approach, the expression of all or part of the high molecular weight subunit of MTP can be assessed immunologically, for example, by immunoblotting with antibody to MTP (Western blotting).
In the fifth approach, expression of the high molecular weight subunit of MTP can be measured by assaying for MTP
enzyme activity using known methods. For example, the assay described herein below may be employed.
In the sixth approach, oligonucleotide primers homologous to sequences present in the expression system (i.e., expression vector sequences or MTP sequences) are used in a PCR to produce a DNA fragment of predicted length, indicating incorporation of the expression system in the host cell.
The DNA sequences of expression vectors, plasmids or DNA molecules of the present invention may be determined by various methods known in the art. For example, the dideoxy chain termination method as described in Sanger gt,a(., p1oc. Nati. Acad.
Sci. USA 7A, 5463-7 (1977), or the Maxam-Gilbert method as described in proc. Natl. Acad. Sci. USA 7A, 560-4 (1977) may be employed.
In order to express catalytically active MTP, it may be necessary to produce a protein complex containing both the high and low molecular weight subunits of MTP. The low molecular DC21 a weight subunit of MTP is the previously characterized protein, protein disulfide isomerase (PDI). PDI cDNAs have been cloned from human [Pihlajaniemi 6t al., EMBO J. 1, 643-9 (1987)], bovine [Yamaguchi 2LQL, 8iochem. Biophys. Res. Comm. L4fi, 1485-92 (1987)], rat [Edman et a1., Nature 3.1Z, 267-70 (1985)] and chicken [Kao et a1., Connective Tissue Research ]ft, 157-74 (1988)].
Various approaches can be used in producing a protein containing both the high and low molecular weight subunits of MTP. For example, cDNA sequences encoding the subunits may be inserted into the same expression vector or different expression vectors and expressed in an appropriate host cell to produce the protein.
It should, of course, be understood that not all expression vectors and DNA regulatory sequences will function equally well to express the DNA sequences of the present invention. Neither will all host cells function equally well with the same expression system. However, one of ordinary skill in the art may make a selection among expression vectors, DNA regulatory sequences, and host cells using the guidance provided herein without undue experimentation and without departing from the scope of the present invention.

Polypeotides The present invention further concerns polypeptide molecules comprising all or part of the high molecular weight subunit of MTP, said polypeptide molecules preferably having all or part of the amino acid sequence as shown in SEQ. ID. NOS. 3, 4, or 3 together with 6. In the case of polypeptide molecules comprising part of the high molecular weight subunit of MTP, it is preferred that polypeptide molecules be at least about 5 to 8 sequential amino acids in length, more preferably at least about 15 to 20 sequential amino acids in length. Also preferred are polypeptides at least about 180 sequential amino acids in length, DC21 a which may approximate the size of a structural domain within the protein. -All amino acid sequences are represented herein by formulas whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus.
The polypeptides of the present invention may be obtained by synthetic means, i.e., chemical synthesis of the polypeptide from its component amino acids, by methods known to those of ordinary skill in the art. For example, the solid phase procedure described by Houghton gtaõ Proc. Natl. Acad. Sci. A2, 5131-5 (1985) may be employed. It is preferred that the polypeptides be obtained by production in prokaryotic or eukaryotic host cells expressing a DNA sequence coding for all or part of the high molecular weight subunit of MTP, or by in vitro translation of the mRNA encoded by a DNA sequence coding for all or part of the high molecular weight subunit of MTP. For example, the DNA
sequence of SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7 and 8 or any part thereof may be synthesized using PCR as described above and inserted into a suitable expression vector, which in turn may be used to transform a suitable host cell. The recombinant host cell may then be cuftured to produce the high molecular weight subunit of MTP.
Techniques for the production of polypeptides by these means are known in the art, and are described herein.
The polypeptides produced in this manner may then be isolated and purified to some degree using various protein purification techniques. For example, chromatographic procedures such as Ion exchange chromatography, gal filtration chromatography and immunoaffinity chromatography may be employed.
The polypeptides of the present invention may be used in a wide variety of ways. For example, the polypeptides may be used to prepare in a known manner polyclonal or monoclonal DC21 a antibodies capable of binding the polypeptides. These antibodies may in turn be used for the detection of the polypeptides of the present invention in a sample, for example, a cell sample, using immunoassay techniques, for example, radioimmunoassay, enzyme immunoassay, or immunocytochemistry. The antibodies may also be used in affinity chromatography for isolating or purifying the polypeptides of the present invention from various sources.
The polypeptides of the present invention have been defined by means of determined DNA and deduced amino acid sequencing. Due to the degeneracy of the genetic code, other DNA sequences which encode the same amino acid sequences depicted in SEQ. ID. NOS. 3, 4, 3 together with 6,-or-any part thereof may be used for the production of the polypeptides of the present invention.
It should be further understood that alleiic variations of these DNA and amino acid sequences naturally exist, or may be intentionally introduced using methods known in the art. These variations may be demonstrated by one or more amino acid changes in the overall sequence, such as deletions, substitutions, insertions, inversions or addition of one or more amino acids in said sequence. Such changes may be advantageous in producing or using the polypeptides of the present invention; for example in isolation of MTP or the polypeptides by affinity purification. Amino acid substitutions may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphiphathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine, glycine, alanine; asparagine, glutamine; serine, _ threonine; phenylaianine, tyrosine. Other contemplated variations DC21 a include salts and esters of the aforementioned poiypeptides, as well as precursors of the aforementioned polypeptides; for example, precursors having N-terminal substituents such as methionine, N-formylmethionine and leader sequences. All such variations are included within the scope of the present invention.
Method for detection of nucleic acids The present invention further conc rns a method for detecting a nucleic acid sequence coding for all or part of the high molecular weight subunit of MTP or a related nucleic acid sequence, comprising contacting the nucleic acid sequence with a detectable marker which binds specifically to at least a portion of the nucleic acid sequence, and detecting the marker so bound.
The presence of bound marker indicates the presence of the nucleic acid sequence. Preferably, the nucleic acid sequence is a DNA sequence having all or part of the nucleotide sequence substantially as shown in SEQ. ID. NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7 and 8, or is complementary thereto.
A DNA sample containing the DNA sequence can be isolated using various methods for DNA isolation which are well-known to those of ordinary skill in the art. For example, a genomic DNA sample may be isolated from tissue by rapidly freezing the tissue from which the DNA is to be isolated, crushing the tissue to produce readily digestible pieces, placing the crushed tissue in a solution of proteinase K and SDS, and incubating the resulting solution until most of the cellular protein is degraded. The genomic DNA is then deproteinized by successive phenoVchloroform/isoamyl alcohol extractions, recovered by ethanol precipitation, and dried and resuspended in buffer.
Also preferred is the method in which the nucleic acid sequence is an RNA sequence. Preferably, the RNA sequence is an mRNA sequence. Additionally preferred is the method in which 20911Q?
DC21 a the RNA sequence is located in the cells of a tissue sample. An RNA sample containing the RNA sequence may be isolated using various methods for RNA isolation which are well-known to those of ordinary skill in the art. For example, an RNA sample may be isolated from cultured cells by washing the cells free of medium and then lysing the cells by placing them in a 4 M guanidinium solution. The viscosity of the resulting solution is reduced by drawing the lysate through a 20-gauge needle. The RNA is then pelleted through a cesium chloride step gradient, and the supernatant fluid from the gradient carefully removed to allow complete separation of the RNA, found in the pellet, from contaminating DNA and pnatein.
The detectable marker useful for detecting a nucleic acid sequence coding for all or part of the high molecular weight subunit of MTP or a related nucleic acid sequence, may be a labeled DNA sequence, including a labeled cDNA sequence;
having a nucleotide sequence complementary to at least a portion of the DNA sequence coding for all or part of the high molecular weight subunit of MTP.
The detectable marker may also be a labeled RNA having a sequence complementary to at least a portion of the DNA
sequence coding for all or part of the high molecular weight subunit of MTP.
The detectable markers of the present invention may be labeled with commonly employed radioactive labels, such as 32P
and 35S, although other labels such as biotin or mercury may be employed. Various methods well-known to those of ordinary skill in the art may be used to label the detectable markers. For example, DNA sequences and RNA sequences may be labeled with 32P or 35S using the random primer method.
Once a suitable detectable marker has been obtained, various methods well-known to those of ordinary skill in the art may be employed for contacting the detectable marker with the sample of interest. For example, DNA-DNA, RNA-RNA and DNA-DC21 a RNA hybridizations may be performed using standard procedures known in the art. In a typical DNA-DNA hybridization procedure for detecting DNA sequences coding for all or part of MTP in genomic DNA, the genomic DNA is first isolated using known methods, and then digested with one or more restriction enzymes.
The resulting DNA fragments are separated on agarose gels, denatured jasftu, and transferred to membrane filters. After prehybridization to reduce nonspecific hybridization, a radiolabeled nucleic acid probe is hybridized to the immobilized DNA fragments. The membrane is then washed to remove unbound or weakly bound probe, and is then autoradiographed to identify the DNA fragments that have hybridized with the probe.
The presence of bound detectable marker may be detected using various methods well-known to those of ordinary skill in the art. For example, if the detectable marker is radioactively labeled, autoradiography may be employed. Depending on the label employed, other detection methods such as spectrophotometry may also be used.
It should be understood that nucleic acid sequences related to nucleic acid sequences coding for all or part of the high molecular weight subunit of MTP can also be detected using the methods described herein. For example, a DNA probe that has conserved regions of the gene for the high molecular weight subunit of human or bovine MTP can be used to detect and isolate related DNA sequences (e.g., a DNA sequence coding for the high molecular weight subunit of MTP from mice, rats, hamsters, or dogs). All such methods are included within the scope of the present invention.

Methods for detectina MTP inhibitors The present invention further concerns methods for detecting inhibitors of MTP. In particular, the present invention concerns a process for detecting an inhibitor of MTP comprising:
(a) incubating a sample thought to contain an inhibitor of MTP with DC21 a detectably labeled lipids in donor particles, acceptor particles and MTP; and (b) measuring the MTP stimulated transfer of the detectably labeled lipids from the donor particles to the acceptor particles. In this assay, an inhibitor would decrease the rate of MTP-stimulated transfer of detectable labeled lipid from donor to acceptor particles. The detection may be carried out by nuclear magnetic resonance (NMR), electron spin resonance (ESR), radiolabeling (which is preferred), fluorescent labeling, and the like. The donor and acceptor particles may be membranes, HDL, low density lipoproteins (LDL), SUV, lipoproteins and the like.
HDL and SUV are the preferred donor particles; LDL and SUV are the preferred acceptor particles.
The foregoing procedure was carried out to identify the MTP
inhibitor A

\
N N / `
O
which has the name 2-[1-(3, 3-diphenytpropyl)-4-piperidinyl]-2, 3-dihydro-3-oxo-1 jj isoindote hydrochloride (herein referred to as "compound A"). Preparation of compound A is described in U. S.
Pat. No. 3, 600, 393. The foregoing procedure also identified the MTP inhibitor B
O

N
N ~ OCH3 F
which has the name 1-[3-(6-fluoro-l-tetralanyl)methyl]-4-0-methoxyphenyl piperazine (herein referred to as "compound B").

209i10194 DC21 a These compounds were identified by the procedures described in the working examples hereinafter. -Methods of treatment The present invention also concerns a novel method for preventing, stabilizing or causing regression of atherosclerosis in a mammalian species comprising administration of a therapeutically effective amount of an agent which decreases the amount or activity of MTP.
The present invention further concerns a novel method for lowering serum lipid levels, such as cholesterol or TG levels, in a mammalian species, which comprises administration of a therapeutically effective amount of an agent which decreases the amount or activity of MTP.
The treatment of various other conditions or diseases using agents which decrease the amount of activity of MTP is also contemplated by the present invention. For example, agents which decrease the amount or activity of MTP and therefore decrease serum cholesterol and TG levels, and TG, fatty acid and cholesterol absorption are likely to be useful in treating hypsrcholesterolemia, hypertriglyceridemia, hyperlipidemia, pancreatitis, hyperglycemia and obesity.
Various agents which effectively decrease the amount or activity of MTP can be used in practicing the methods of the present invention. MTP inhibitors can be isolated using the screening methodology described hereinabove and in Example 5 hereinbelow. Compounds such as A and B, which are identified as inhibitors of MTP (see Example 6 hereinbelow), are useful in specific embodiments of the foregoing methods of treatment.
Antisense molecules may be used to reduce the amount of MTP. [Sea, Toulme and Helene, Bene Z?., 51-8 (1988); Inouye, Gene, Z?., 25-34 (1988); and Uhlmann and Peyman, Chern*cal Reviews M, 543-584 (1990)]. MTP antisense molecules can be designed based on the foregoing genomic DNA and cDNA, DC21 a corresponding 5' and 3' flanking control regions, other flanking sequences, or intron sequences. Such antisense molecules include antisense oligodeoxyribonucleotides, oligoribonucleotides, oligonucleotide analogues, arid the like, and may comprise about 15 to 25 bases or more. Such antisense molecules may bind noncovalently or covalently to the DNA or RNA for the high molecular weight subunit of MTP. Such binding could, for example, cleave or faciltate cleavage of MTP DNA or RNA, increase degradation of nuclear or cytoplasmic mRNA, or inhibit transcription, translation, binding of transactivating factors, or pre-mRNA splicing or processing. All of these effects would decrease expression of MTP and thus make the antisense molecules useful in the foregoing methods of treatment.
Potential target sequences for an antisense approach include but are not limited to the DNA or RNA sequence encoding MTP, its 5' and 3' flanking control regions, other flanking sequences, and nonclassic Watson and Crick base pairing sequences used in formation of triplex DNA. Antisense molecules directed against tandem sequences for the high molecular weight subunit of MTP may be advantageous.
Antisense molecules may also contain additional functionalities that increase their stability, activity, transport into and out of cells, and the like. Such additional functionalities may, for example, bind or facilitate binding to target molecules, or cleave or facilitate cleavage of target molecules.
Vectors may be constructed that direct the synthesis of antisense DNA or RNA. In this case, the length of the antisense molecule may be much longer; for example, 400 bp.

Demonstration of relationship between MTP and serum cholesterol levels, TG levels, and atherosclerosis The methods of the present invention for lowering serum cholesterol or TG levels or preventing, stabilizing or causing regression of atherosclerosis are based in part on the discovery by DC21 a the inventors that the genetic disease abetalipoproteinemia is caused by a lack of functional MTP. The inventors have demonstrated a gene defect in two abetalipoproteinemic subjects by the following methods.
Assay for TG transfer activity In abetaiipoproteinemic subjects A. MTP Assay TG transfer activity was measured as the protein-stimulated rate of TG transfer from donor SUV to acceptor SUV. To prepare donor and acceptor vesicles, the appropriate lipids in chloroform were mixed in a 16 x 125 mm borosilicate glass screw cap tube (Fisher Scientific Co., Pittsburg, PA, Cat. no. 14-933-1 A) and then dried under a stream of nitrogen. Two mL 15/40 buffer (15 mM
Tris, pH 7.4, 40 mM sodium chloride, 1 mM EDTA, and 0.02%
NaN3 ) were added to the dried lipids. (or 100 L per assay, which ever is the least volume), a stream of nitrogen was blown over the buffer, then the cap was quickly screwed on to trap a nitrogen atmosphere over the lipid suspension. Lipids in the buffer were bath-sonicated in a Special Ultrasonic Cleaner (Cat. no.
G112SP1, Laboratory Supplies Co., Hicksville, NY). The donor and acceptor phosphatidytcholine (PC) (egg L-alpha-phosphatidylcholine, Sigma Chem. Co., St. Louis, MO) was radiolabeled by adding traces of [3H] dipalmitoyl-phosphatidylcholine (phosphatidyicholine L-alpha-dipaimitoyl [2-paimitoyi-9,10, 3H (N)], 33 Ci/mmoi, DuPont NEN) to an approximate specific activity of 100 cpm/nmol. Donor vesicles containing 40 nmol egg PC, 0.2 mol% [14C]TG [mixture of labeled (triolein [carboxyl-14C]-, about 100 mCi/mmol, DuPont NEN) and unlabeled (triolein,. Sigma Chem. Co., St. Louis, MO) triolein for a final specific activity of about 200,000 cpm/nmot], and 7.3 mol%
cardiolipin (bovine heart cardiolipin, Sigma Chemical Co.) and acceptor vesicles containing 240 nmol egg PC and 0.2 mol% TG
were mixed with 5 mg fatty acid free bovine serum albumin (BSA) DC21 a and an aliquot of the MTP samples in 0.7 to 0.9 mL 15/40 buffer and incubated for 1 hour at 37 C. The transfer reaction was terminated by the addition of 0.5 mL DEAE-cellulose suspension (1:1 suspension DE-52, preswolien DEAE-cellulose anion exchange, Fisher, Cat. no. 05720-5 to 15 mM Tris, pH 7.4, 1 mM
EDTA, and 0.02% NaN3). The reaction mixture was agitated for 5 minutes and the DEAE-cellulose with bound donor membranes (the donor membranes contained the negatively charged cardiolipin and bound to the DEAE) were sedimented by low speed centrifugation.
The 14C-TG and 3H-PC remaining in the supernatant were quantitated by scintillation counting. TG transfer was calculated by comparing the ratio of 14C-TG (transferred from the donor membranes to the acceptor membranes) to 3H-PC (a marker of acceptor vesicle recovery) present in the supernatant following a transfer reaction to the ratio of total donor14C-TG to acceptor [3H]PC in the assay before the transfer reaction. The percentage of 14C-TG transfer was calculated as follows:

% TG Transfer = (14C-TG/3H-PC)SU x 100%
( C-TGd0n/ H-PCaco)total To calculate the MTP-stimulated rate of TG transfer, the TG
transfer rate in the absence of MTP was subtracted from the TG
transfer rate in the presence of MTP. First order kinetics was used to calculate total TG transfer.

B. Antibody Production Anti-88 kDa antibodies were obtained from the University of Cincinnati. The production of anti-88 kDa has been previously described. Wetterau gLaI,, J. Biol. Chem. M, 9800-7 (1990). To help address the specificity of the anti-sera in human intestinal homogenates, affinity purified anti-88 kDa was generated. Eight to 10 mg of purified MTP.was dialyzed into 0.1 M MOPS, pH 7.5 and then added to 4 mL Bio Rad Affigel 15 (Bio-Rad, Richmond, CA) DC21 a which had been prewashed 3 times with water at 4 C. The MTP
was allowed to couple to the matrix at room temperature for two hours and then it was placed at 4 C overnight. The remaining reactive sites on the affigel were blocked by the addition of 0.1 mL
1 M ethanolamine, pH 8.0, per mL gel. Optical density measurements of eluted protein were performed according to the manufacturer's instructions and indicated that more than 90% of the MTP was coupled to the column. The column was washed with 50 mL 10 mM Tris, pH 7.5 followed by 50 mL 100 mM glycine, pH 2.5, followed by 50 mL 10 mM Tris, pH 8.8, followed by 50 mL
100 mM triethylamine, pH 11.5, and finally the column was reequilibrated in 10 mM Tris, pH 7.5.
The antibodies in the antiserum were partially purified by ammonium sulfate precipitation (226 mg ammonium sulfate per mL serum). The pellet was resuspended and dialyzed into 15 mm, Tris, pH 7.5, 1 mM EDTA, 0.02% sodium azide, and 150 mM
sodium chioride. The partially purified antibodies were slowly applied to the MTP-affigel column over a two-hour period (the antibodies were cycled through the column three times). The column was washed with 100 mL 10 mM Tris, pH 7.5, followed by 100 mL 10 mm, Tris, pH 7.5, 500 mM sodium chloride, followed by 50 mL 100 mM glycine, pH 2.5 (this fraction was collected into 5 mL of 1M Tris, pH 8.0), followed by 10 mM Tris, pH 8.8 until the column was at neutral pH, followed by 50 mL triethylamine pH
11.5 (this fraction was collected into 5 mL 1M Tris, pH 8.0), and finally the column was reequilibrated with 10 mM Tris, pH 7.5.
Antibodies which eluted in the acidic wash were retained and used for immunoblot analysis of protein fractions.

C. Western Blot with anti-88 kDa Antibodies To confirm the specificity of the antibodies and to detect the 88 kDa component of MTP in tissue homogenates, purified bovine MTP or the fraction to be tested were fractionated by SDS-PAGE
[essentially as described by Laemmli, Nature 22Z, 680-5 (1970)]

DC21 a using a 0.75 mm Hoeffer Scientific Instrument Gel Apparatus (San Francisco, CA). The protein was then transferred to nitrocellulose by Westem blotting using a BioRad Trans-blot cell (Bio-Rad, Richmond, CA). The blotting buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol) was precooled to 4 C. The proteins were transferred for 100 minutes at 250 milliamperes at room temperature. The membranes were blocked 5-10 minutes with blocking buffer (400 L antifoam, about 10 mg of thimersal, and 200 g nonfat dry milk in 4 liters 50 mM Tris, pH 7.7, 150 mM
sodium chloride). An aliquot of the antiserum (1:300 dilution) or affinity purified antibody (1:25 dilution of affinity-purified antibodies) was added and allowed to react overnight at room temperature. Following washing with blocking buffer, the secondary antibody, goat anti-rabbit IgG coupled to horseradish peroxidase (BioRad), was added at a dilution of 1:2000 and allowed to react for 3 hours at room temperature. Following a washing step, the secondary antibody was visualized with developer, 50 mg imidizale, 50 mg 3,3'diaminobenzidine tetrahydrochloride, and 50 L H202 (30% solution) in 50 mL
blocking buffer.

D. MTP in Intestinal Biospies Intestinal biopsies from fasted control and disease state subjects were frozen and shipped to Bristol-Myers Squibb, Princeton on dry ice. Biopsies were homogenized with a polytron (Polytron PT3000, Brinkmann Instrument, Inc., Westbury, NY) at 1/2 maximal settting. Typically, one biopsy was homogenized in 0.25 mL homogenization buffer (50 mM Tris, pH 7.4, 50 mM KCI, 5 mM EDTA, 5 g/mL leupeptin, and 2 mM PMSF). An aliquot of the protein was adjusted to 0.7 mL and 1.4% SDS and the protein concentration was measured by the method of Lowry gla,. [J. Bio1.
Chem. 10, 265-75 (1951)]. The homogenate was diluted with homogenization buffer to about 1.75 mg protein/mL. In some cases, the protein was already more dilute and was used directly.

DC21 a To release the soluble proteins from the microsomal fraction, one part deoxycholate solution (0.56%, pH 7.5) was addectto 10 parts diluted homogenate with vortexing. The sample was incubated at 4 C for 30 minutes, then centrifuged at 103,000 x g for 60 minutes.
The supernatant was removed, diluted 1:1 with 15/40 buffer, and then dialyzed ovemight into 15/40 buffer. Aliquots of the treated biopsies were assayed for TG transfer activity and Westem blot analysis was used to detect 88 kDa protein. TG transfer activity was expressed as the percentage of donor TG transferred per hour as a function of homogenized intestinal biopsy protein.
E. Results with AbetalipQoroteinemic Subjects To investigate whether there is a relationship between defective MTP and abetalipoproteinemia, MTP activity in duodenal or duodenal-jejunal biopsies was measured from five control subjects and four abetatipoproteinemic subjects having the classic genetically recessive form of abetalipoproteinemia. Intestinal biopsies from the five normal subjects were homogenized and treated with detergent as described hereinabove. TG transfer activity was readily detectable in biopsies from all five subjects (Figure 2).
The TG transfer activity in the biopsies was further characterized. To confirm that TG hydrolysis was not interfering with lipid transfer activity measurements, one subject's acceptor vesicles (which contained the transported lipid) were extracted after the transfer reaction, and the identity of the 14C-TG was confirmed by thin layer chromatography. All of the 14C-TG had a mobility identical to that of authentic TG, confirming that intact TG
was being transported in the assay.
The human MTP was characterized for its heat stability. It was inactivated when heated to 60 C for 5 minutes. The loss of activity demonstrates that the lipid transfer activity being measured was not from an intracellular form of the cholesteryl ester transfer DC21 a protein (CETP), which is heat-stable under these conditions. lhm et al., J. Biol. Chem. JU, 4818-27 (1982). -Intestinal biopsies from four abetalipoproteinemic subjects were obtained, homogenized, and TG transfer activity was measured as desribed herein above. No transfer activity was recovered from the biopsies of any of the four subjects (Figure 3).
The lack of detectable TG transfer activity could have been related to an inability to release MTP from the microsomes of the abetalipoproteinemic biopsies by deoxycholate treatment. To test this possibility, the microsomes from one subject were sonicated in addition to being treated with detergent. Bath sonication independently releases TG transfer activity comparable to that of detergent treatment. Even under these conditions, no TG transfer activity was detectable.
The next possibility considered was that the lack of detectable TG transfer activity was related to the inability to detect it in cells which contain large intracellular fat droplets such as those which occur in abetalipoproteinemia. To test this possibility, three controls were run. First, TG transfer activity was measured from a biopsy of a subject with chylomicron retention disease.
Subjects with chylomicron retention desease have a defect in the assembly or secretion of chylomicrons and have large fat droplets in their enterocytes, analogous to abetalipoproteinemic subjects.
In addition, TG transfer activity was measured from a biopsy taken from an individual who was not fasted prior to the biopsy and from a homozygous hypobetalipoproteinemic subject. Both these subjects also had fat-filled enterocytes. In all three cases, TG
transfer activity comparable to that of the normal subjects was found (Figure 4), confirming that the presence of intracellular lipid droplets does not interfere with our ability to recover and detect TG
transfer activity.
To establish the biochemical defect responsible for the absence of transfer activity, the soluble proteins following release of MTP from the microsomal fraction of the homogenized biopsy DC21 a were analyzed by Western blot analysis with antibodies raised against the 88 kDa component of bovine MTP. When normal (Figure 5) or control (Figure 6) subjects were examined with a polyclonal anti-88 kDa antibody, a band comparable to that of the 88 kDa component of bovine MTP was observed. In addition, additional proteins of increased mobility also cross-reacted with this antibody. To confirm the identity of the 88 kDa component of human MTP, the antibody was af1"inity-purified on an MTP affinity column. Following this'treatment, only the protein of molecular weight comparable to that of the 88 kDa component of bovine MTP
was immunoreactive (Figure 7).
Western blot analysis of the soluble proteins following detergent treatment of the microsomes of all five normal subjects and three control subjects demonstrated the presence of the 88 kDa component of MTP (Figures 5 to 7). In contrast, no protein corresponding to the 88 kDa component of bovine MTP was apparent in the abetalipoproteinemic subjects (Figure 8). In addition, a similar analysis was performed with 100 g protein from the whole intestinal homogenates from two abetalipoproteinemic subjects. Again, no band corresponding to the 88 kDa component of MTP was apparent (Figure 8). As a control, immunoblot analysis with anti-PDI antibodies demonstrated the presence of PDI in the latter two abetalipoproteinemic subjects. These resufts demonstrate that the biochemical basis for the absence of MTP activity in the abetalipoproteinemic subjects is the marked deficiency or the absence of the 88 kDa component of MTP.

Demonstration of a gene defect In an abetalipoproteinemic subject Amplification of mRNA and DNA by PCR
Two intestinal biopsies were obtained from the duodenal mucosa of a 39-year-old abetalipoproteinemic patient. Previous analysis demonstrated that neither MTP activity nor the 88 kDa component of MTP were detectable in intestinal biopsies taken from this subject. Each biopsy weighed 5-10 mg and-was stored frozen at -70 C. To isolate total RNA, one frozen biopsy was placed into a microfuge tube containing 0.8 mL of cold RNAzol B
(CinnaBiotecx labs, Friendswood, Texas). The biopsy was homogenized immediately by polytron (Brinkmann, Westbury, NY) for 6 strokes on setting 10. Chloroform (80 L) was added and the mixture inverted gently 20 times. After a 5-minute incubation on ice, the mixture was centrifuged at 14,000 rpm in an Eppendorf microfuge 5415 (Brinkmann) for 15 minutes at 4 C. Total RNA was precipitated by adding 350 L isopropanol to the supematant. The yield from the biospy was 20 g of total RNA, or about 2 g RNA
per mg of tissue (0.2%). -RNA (50 ng) was reverse transcribed into first strand cDNA
using 2.5 M random hexamer primers, 5 mm, magnesium chloride, 1 mM each deoxynucleotide triphosphate (dNTP), 1 U/ L
RNAsin, 2.5 U/ L Moloney Murine Leukemia Virus reverse transcriptase ((M-MLV-RT), and 1 X PCR reaction buffer (Perkin-Elmer-Cetus RNA-PCR kit No. N808-0017). The 20 L reaction was incubated at room temperature for 10 minutes to anneal the primers, and then at 42 C for 30 minutes to reverse transcribe the RNA. The reaction was terminated by heating to 99 C for 5 minutes and cooling to 5 C. The first strand cDNA was added to a 100 L PCR containing 0.15 M forward and reverse primers, 2 mM magnesium chloride, 0.2 mm, each dNTP, and 2.5 U Taq polymerase in 1.25X PCR buffer. Amplification was conducted in a Perkin-Elmer GeneAmpTM PCR System 9600 model thermal cycler for 50 cycles consisting of 94 C for 30 seconds, 50 C for 30 seconds, and 72 C for 1 minute. The reaction was then incubated at 72 C for 7 minutes. The forward and reverse primers used to amplify the 5' region of the RNA encoding the 88 kDa component of MTP are shown below, 5' to 3'.

DC21a Forward Primers Seauence SEO. ID. NO.

Reverse Primers Secruence SEO. ID. NO.

1029R CGCfGLATCCTTCTGACAGCCTCAGCCTTGGA 15 2117R CGGCGGA=AGCATAGGAGTCAAGGTTCTC 17 Shown below are the primer combinations used the PCR
product length.
.Primer Pair Product Lenath (bn) 15F + 678R 664 15F + 839R 825 41F + 1029R 998 57SF + 1029R 470 900F + 1588R 689 900F + 2117R 1228 The primer sequences are based on the normal human cDNA encoding the 88 kDa component of MTP. All primers are written 5' to 3'. F refers to the forward primer, and R to the reverse DC21 a primer. The underlining identifies restriction sites recognized by Eco RI (primer 578F) or Bam HI (primers 1029R and 2117R), which were incorporated into the 5' end of the primers.
Subject genomic DNA was isolated from a second frozen intestinal biopsy. The biopsy was placed into a microfuge tube containing 400 L extraction buffer (10 mM Tris.Cl, pH 8.0, 0.1 M
EDTA, 0.5% SDS, 20 g/mL RNAse I) and homogenized immediately. Homogenization was by polytron for 5 strokes at setting 10. Proteinase K was added to a final concentration of 100 g/mi and the reaction incubated at 50 C for 3 hours. The mixture was swiried periodically.
After cooling the reaction to room temperature, 400 L Tris-saturated phenoUchloroform (pH 8.0) was added. The tube was inverted gently for 5 minutes and then centrifuged for 5 minutes at 14,000 rpm at room temperature. 2 M sodium chloride (35 L) and ethanol (0.7 ml) were added to the supernatant (350 u.L) to precipitate the DNA. The DNA was centrifuged briefly, washed gently with 70% ethanol, dried briefly, and resuspended in 20 L
of deionized water (dH2O). The yield of DNA was 20 g, or about 2 g DNA per mg tissue (0.2%).
Genomic DNA (0.5 g) was heated to 95 C for 5 minutes and added immediately to a 100 L PCR reaction containing 0.15 M forward and reverse primers, 2 mM magnesium chloride, 0.2 mM each dNTP, and 2.5 U Taq polymerase in 1.25X PCR buffer (Per(in-Elmer-Cetus). Amplification was conducted in a Perkin-Elmer GeneAmp PCR System 9600 model thermal cycler for 3 cycles consisting of 97 C for 30 seconds, 50 C for 30 seconds, and 72 C for 1 minute. An additional 32 cycles consisting of 94 C
for 30 seconds, 50 C for 30 seconds, and 72 C for 1 minute were run. The reaction was then incubated at 72 C for 7 minutes.
Exon 2 of the gene encodes bases 109-296 of the 88 kDa component of MTP RNA. The primers (SEQ. ID. NOS. 18 and 19) used to amplify exon 2 of the gene encoding the 88 kDa component of MTP from subject genomic DNA are shown below.

DC21 a Primer Pair SEO. ID. NO.

These primers were designed based on the normal human DNA sequence encoding the 88 kDa component of MTP. The primers are complementary to the introns flanking the 188 bp exon 2 so that the entire exon is amplified in the PCR reaction. The amplification product size, including the primers and flanking intronic regions, is 292 bp long.
B. Sequencina of PCR products The PCR products obtained from both RNA- and DNA-PCR
were electrophoresed on a 1.4% agarose gel in TAE buffer (40 mM Tris-acetate, 1 mM EDTA pH 8.0). The gel was stained for 5 minutes in 0.5 mg/mL ethidium bromide in water, and destained in water for 10 minutes. The DNA was visualized on an ultraviolet light box. The bands containing the desired PCR product were excised with a razor blade, and the DNA was purified by the GeneClean method (Bio 101, La Jolla, CA). The DNA was eluted from the silica matrix in 20 L of distilled water. Each PCR
reaction yielded approximately 1 g of the desired DNA fragment.
A portion of the purified DNA was sequenced directly by Taq polymerase cycle sequencing on an Applied Biosystems, Inc., 373 Automatic Sequencer, as described by Tracy and Mulcahy, SiotechniaUes, 11, 68 (1991).
The remaining DNA was prepared for cloning into a plasmid vector by producing blunt-ends with T4 DNA polymerase followed by phosphorylation with T4 polynucleotide kinase. DNA
(500 ng) was added to a 50 L reaction mixture containing 20 ,pd each dNTP, 1 mM ATP, 4.5 units T4 DNA polymerase, 5 units T4 polynucleotide kinase in 50 mM Tris HCI pH 7.5, 10 mM
magnesium chloride, 1 mM dithiothreitol, and 50 g/mL BSA.
Incubation was at 37 C for 1 hour. The DNA was then purified from__ the reaction mixture by GeneClean. The DNA was eluted in 10 L

DC21 a dH2O. The blunt-ended DNA was ligated into pUC1 8 cut previously with Sma I and dephosphorylated (Pharmacia). Dh5a cells (100 L, Gibco-BRL) were transformed according to the protocol supplied by the manufacturer. Plasmid DNA was amplified and isolated by the alkaline lysis procedure described in Molecular Clonina (Sambrook, Fritsch, and Maniatis, eds.) Cold Spring Harbor Laboratory Press, 1.25-1.28 (1989). The plasmid clones were sequenced as described in Example 1.

Besyft8 Direct sequence from three independent RNA-PCR
reactions revealed a deleted cytosine at base 262 of the cDNA
relative to the start site of translation in the abetalipoproteinemic subject. The one base deletion shifts the reading frame and leads to a stop codon (TGA) 21 bases downstream. Translation of the mutant RNA would terminate at amino acid residue 78. Below is a comparison of the normal and the abetalipoproteinemic subject's DNA and deduced amino acid sequences Base 255 287 AGG AAT CCT GAT GGT GAT GAT GAC CAG TTG ATC Normal AA R N P D G D D D Q L I

Base 255 286 AGG AAT C-TG ATG GTG ATG ATG ACC AGT TGA TG Abeta AA R N L M V M M T S STOP
(SEQ. ID. NOS. 20 to 23, respectively).
Direct sequence analysis of 2 independent PCR
amplifications of genomic DNA showed the deletion. This indicates that both alleles of the gene encoding the 88 kDa component of MTP in this subject exhibits the frameshift mutation.
In addition, the DNA fragments were cloned into pUC1 8 for sequencing. Eight plasmid clones also exhibit the deleted cytosine further confirming the frameshift mutation on both alleles.

DC21 a Demonstration of a gene defect in a second abetaiipoproteinemic subject -A. Methods Genomic DNA was isolated from blood from a second abetalipoproteinemic subject using Qiagen (Chatsworth, Ca) kit no. 13343, following the manufacturer's protocol. Like the first subject, we have previously demonstrated that neither MTP activity nor the 88 kDa component of MTP could be detected in intestinal biopsies from this subject. Three tiundred g of this genomic DNA
was sent to Stratagene (La Jolla, CA) to be made into a genomic DNA library in the lambda DASHTM Vector(Stratagene). In addition, a normal genomic library in the lambda DASHTM vector was purchased from Stratagene (catalogue no. 943202).
Two million independent recombinant phage plaques from each library were screened for genomic DNA inserts containing sequences homologous to bovine MTP cDNA. The screening process was similar to that for the cDNA library screen in Example 1 except that the E. coli host strain was PLK 17, hybridization and wash temperatures were at 60 C, and the wash buffer was 1 X
SSC, 0.1 % SDS. The probe for the genomic library screen was the 2.4 kb Eco RI fragment from the bovine cDNA clone no. 22, 32P-labeled exactly as in example 2. Putative positive clones (about 30 from each library) were rescreened and remained positive through two additional rounds of hybridization analysis.
Following the tertiary screen, single, isolated positive plaques were excised from the agar plates and deposited into 1 mL of SM
buffer with 50 L chloroform. Phage titer was amplified for each phage stock following the "Small-scale liquid cuftures" protocol from Sambrook, 91W., supra, p 2.67. One hundred L of the amplified stocks was mixed with 100 L of prepared PLK 17 plating cells and 100 L of 10 mh& magnesium chloride, 10 mm calcium chloride and incubated at 37 C.for 15 minutes. This mixture was then used to inoculate 50 mL 2X NZY (Bethesda Research Laboratories) with 0.2% Casamino Acids (CAA, Fisher DC21 a Scientific no. DF0288-01-2) and grown ovemight at 37 C.
Lambda DNA was isolated from the lysed cultures using the Qiagen kit no. 12543 using Qiagen buffers and protocol.
Direct DNA sequencing of the genomic DNA inserts was performed as described in Example 1 using lambda DNA as template. Oligonucleotides of about 20 bases, complementary to human cDNA sequence, were used as primers for sequencing normal or abetalipoproteinemic genomic clones. Characterization and sequencing of abetalipoproteinemic and normal genomic clones were performed in parallel (see Example 9). Intron-exon boundaries were identified by comparing genomic and cDNA
sequences. Sequencing primers were designed against intron sequences 5' and 3' to each exon and used to confirm intron/exon boundaries by resequencing the boundaries. In addition, the coding sequence of both DNA strands for each exon of at least one abetalipoproteinemia genomic clone was sequenced. DNA
sequence analysis of exon 13 of the abetalipoproteinemic subject revealed a C-to-T point mutation at base 1830 of the human cDNA. This base change introduces a stop codon at a site that normally encodes the amino acid residue Arg595.
The nucleotide sequence around base 1830 encodes a Taq I endonuclease restriction site (TCGA) in the normal DNA
sequence but not in the abetalipoproteinemic subject's DNA
sequence (TTGA). To confirm this nucleotide change and address homozygosity of this aliele, Taq I digests were performed on genomic DNA from a normal control, the abetalipoproteinemic subject and both parents of the abetalipoproteinemic subject.
Genomic DNA was isolated from blood from a normal control, the abetaiipoproteinemic subject and the abetalipoproteinemic subject's mother and father as described above. Ten g of genomic DNA from each sample was digested with 100 units of Taq I (Bethesda Research Laboratories) in 100 L 1 X REact buffer no. 2 (Bethesda Research Laboratories) at 65 C for 5 hours.
Each digestion reaction was spun at 2,000 rpm in an Ultrafree-MC

DC21 a 10,000 NMWL filter unit (no. UFC3 TGC 00 from Millipore) with a molecular weight cut-off of 10,000, for 30 minutes to reduce the reaction volume to 50 L. The restriction digest reactions were then subjected to agarose gel electrophoresis through a 1 % gel in TEA buffer at 20 vofts for 16 hours. The agarose gel was stained with ethidium bromide, photographed, and then transferred to a nitrocellulose membrane by the method of Southem. E.M.
Southem, J. Mol. Biol. 91, 503-17 (1975).
The probe for the Southern hybridization was a PCR
product containing exon 13 and some flanking intron sequences (see SEQ. ID NO.24, below). The PCR was performed using the GeneAmp Kit (Perkin-Elmer, Cetus Industries) components and protocol with 0.3 g normal genomic DNA as template and 10 picomoles each of the forward and reverse primers in a 100 L
reaction volume. The reaction mix was incubated at 97 C for two minutes, then subjected to 30 cycles consisting of 94 C for 30 seconds, 45 C for 30 seconds, and 72 C for 1 minute, followed by one 7-minute incubation at 72 C and storage at 4 C. The amplified DNA was subjected to electrophoresis through agarose as in example I and the expected 302 bp fragment was excised and eluted from the gel. This exon 13 PCR product was then 32P-labeled as in example 2 and used as a probe for the Southern hybridization. Hybridization and wash conditions were as in example 2. The blot was exposed to X-ray film at -80 C for 5 days.
B. @Ssufts A human genomic library was generated from DNA isolated from a second abetalipoproteinemic subject. Two million phage were probed with a bovine cDNA probe and thirty phage with human genomic DNA inserts homologous to the bovine MTP
cDNA were characterized.
DNA sequence analysis of the genomic DNA inserts from the abetalipoproteinemic subject revealed a C-to-T point mutation at base 1830 in exon 13 of the human MTP gene (exon 13 DC21 a corresponds to bases 1817 to 1914 of the human cDNA). This C-to-T point mutation changes the normal CGA arginine codon at residue 595 to a TGA translational stop signal, resuiting in a 300 amino acid truncation of this protein. This nucleotide change was found on all four independent genomic DNA inserts characterized from this individual.
Shown below is the position of the C-to-T mutation in exon 13 of an abetalipoproteinemic subject. The 302 base DNA
sequence of the normal exon 13 with flanking intron sequence is shown. DNA corresponding to the forward (-->) and reverse (<--) PCR primers used to make the probe for the Southern hybridization are indicated above the appropriate arrows.
Hori2ontal fines represent the intron/exon boundaries. The Taq I
recognition sequence is boxed. An asterisk (*) designates base 1830, the site of the C-to-T mutation.
SEQ. ID. NO. 24.

INTRON EXON

RR~GTRTGTR CRCCRRRRRG RGGTTCTCCT TCCRTRCCCCLRCRflCTTRGC 250 EXON INTRON
RTTGCTGGRR CTGCTRTTRR RTTRCRGTTR TRGTqIGTCR TCRGGTRGTC 300 DC21 a The normal nucleotide sequence surrounding the C at base 1830 (TCGA) encodes a Taq I restriction site. In this -abetalipoproteinemic subject, the sequence at this site is mutated (TTGA). Therefore, Taq I should cut exon 13 at this site in normal DNA, but not in DNA which contains the mutation. There is only one Taq I site in the normal exon 13.
A Southern blot confirms this nucleotide change (Figure 9). The genomic DNA isolated from a control subject, the abetalipoproteinemic subject, and the subject's mother and father was cut to completion with Taq I and probed with sequences from exon 13. The normal DNA is cut by Taq I into two pieces which hybridize to exon 13; the abetalipoproteinemia DNA is not cut with Taq I, evidenced by only one hybridizing band. This result confirms the lack of a Taq I recognition sequence. The DNA from both parents exhibits a mixed pattern, demonstrating the presence of one normal allele and one mutated aliele.

C. Bpalyai;i The foregoing resuits and the conclusions drawn from them can be summarized as follows.
MTP activ'ity and protein are undetectable in the abetalipoproteinemic subjects studied. Mutations in the MTP gene fully explain the lack of protein and activity. Previous results demonstrate that abetalipoproteinemia is a monogenetic disease Kane & Havel,.supr3. From these resufts, one can conclude that abetalipoproteinemia is caused by a loss of MTP activity.
These results demonstrate that MTP activity Is required for the efficient assembly and secretion of lipoprotein particles which contain apolipoprotein B. Loss of MTP activity results in lower serum levels of cholesterol, triglycerides, phospholipids, and cholesterol esters. One can thus conclude that a decrease in the amount of activity of MTP will resuft in lower serum lipid levels.
Moreover, lower serum lipid levels are associated with prevention, stabilization, or regression of atherosclerosis. As DC21 a discused above, loss of the amount or activity of MTP results in lower serum lipid levels. In addition, abetalipoproteinemic subjects lack atherosclerosis. Schaefer, supra; Dische and Porro, Am. J. Med., 4,q, 568-71 (1970); and Sobrevilia gU(., Am. J. Med., 3Z, 821 (1964). One can thus also conclude that inhibition of MTP
wi{I result in the prevention, stabilization, or regression of atherosclerosis.
The following examples further illustrate the present invention. These examples are not intended to limit the scope of the present invention, and may provide further understanding of the invention.

2091i0?

DC21 a Exam l Isolation and DNA Sequence Analysis of- cDNA
Clones Encoding the 88 kDa Component of the Bovine MTP
A commercially available bacteriophage lambda gt10/bovine small intestine cDNA library was purchased from Clontech. 1 X 106 independent recombinant phage plaques were screened for the cDNA corresponding to the 88 kDa component of bovine MTP.
An E. coll bacteria host, strain C600 (Clontech), was prepared for phage infection by growing ovemight to saturation at 30 C in 50 mL of Luria Broth (LB = 10 g sodium chloride, 10 g Bacto-Tryptone and 5 g Yeast Extract per liter) supplemented with 0.2% maRose and 10 mm, magnesium sulfate. The cells were pelleted by low speed centrifugation, resuspended in 20 mL of 10 mM magnesium sulfate and stored at 4 C. Twenty aliquots each of 50,000 phage and 300 L of the C600 cells were incubated at 37 C for 15 minutes, mixed with 7 mL LB + 0.7% agarose and plated on 132 mm LB Plates. The plates were incubated for 7-10 hours at 37 C until distinct phage plaques appeared, then transferred to 4 C.
Duplicate plaque transfers to nitrocellulose membranes were performed for each plate as follows. A nitrocellulose membrane (Schleicher & Schuell, Keene, NH) was placed directly on the phage for 1 minute (first transfer) or 3 minutes (second transfer). The phage DNA adhering to the membrane was then denatured for 1 minute in 0.5 bL sodium hydroxide, 1.5 M sodium chloride, neutralized for 1 minute in 1 M Tris, pH 8.0, 1.5 M sodium chloride, and finally washed for 1 minute in 2 X SSC (1 X SSC =
0.15 M sodium chlorfde, 0.015, M sodium citrate, pH 7.0). The DNA was then permanently fixed onto the nitrocellulose membrane by baking in an 80 C vacuum oven for 2 hours.
The isolation of bovine MTP, including the 88 kDa component, has been previously described. ~, Wetterau and Zilversmit, Chem. Phys. Lin ds 31205-72 (1985); Wetterau et al..
J. Biol. Chem. Za 9800-7 (1990). The sequences of-intemal peptides of the 88 kDa component were used to design oligonucleotides which would hybridize to cDNA that encodes the protein. Bp& Lathe, R., J. Mol. Biol. JM, 1-12 (1985).
The procedures described herein employed probes having the following DNA sequences (listed 5' to 3'):
Probe Seauence SEQ. !D. NO.

T C G G G

Probe 2A is a mixture of thirty-two twenty base oligonucleotides, each encoding the amino acid sequence of the peptide from which this probe was designed. Probe 37A is a unique 33 base sequence and probe 19A is a unique thirty-mer. These oligonucleotide sequences encode amino acid sequences that correspond to intemal peptides.
Oiigonucieotides were obtained from commercial sources as indicated herein or synthesized on a Milligen/Biosearch (Millipore Corp., Bedford, MA) 8700 DNA Synthesizer using beta-cyanoethyl phosphoramidite chemistry. Sequencing primers were desaited on NAP-10 columns (Pharmacia LKB Biotechnologies, Inc., Piscataway, NJ) prior to use. Probes were purified on NENSORB Prep Resin (DuPont Company, NEN Research Products, Boston, MA).
Probe 2A was purchased from Genosys Biotechnologies, Inc. (The Woodlands, Texas) and was labeled by incubating 1 g of the oligonucleotide in 50 mh& Tris-CI, pH 7.5, 10 mh&
magnesium chloride, 5 mh& dithiothreitol (DTT), 0.1 mbA
ethylenediaminetetraacetate (EDTA), and 0.1 mm, spermidine with 10 units T4 polynucleotide kinase and 120 Ci of gamma labeled 32P-ATP in a 50 L reaction volume at 37 C for 30 minutes followed by heat inactivation of the kinase at 68 C for 5 minutes.
Unreacted ATP was removed utilizing a G-25 SephadexTM spin DC21 a column (Boehringer Mannheim Corp., Indianapolis, IN) following the manufacturer's instructions. The labeled oligonucleotide had a specific activity of approximately 2 X 1.08 dpm/ g.
The nitrocellulose membranes were prehybridized for 2 hours at 37 C in 150 mL of hybridization buffer (6 X SSC, 20 mM
NaPO4, 2 X Denhardts, 0.1% SDS, and 100 g/mL salmon sperm DNA) ($ig, Sambrook et a1., supra, p. B15 for Denhardts). The hybridization buffer was replaced and the labeled oligonucleotide probe 2A was added and allowed to hybridize overnight at 37 C.
The membranes were washed in 1 liter of 2 X SSC, 0.1% SDS at 40 C, air-dried, and exposed to Kodak XAR-5 X-ray film for 5 days at -80 C, with a Dupont lightening plus intensifying screen (Dupont, NEN).
Putative positive clones (40) were rescreened with the same probe through two subsequent rounds of hybridization.
Agar plugs corresponding to positive signals on the X-ray films were excised from the original plates and placed in 1 mL SM + 5%
CHCI3 (SM = 5.8 g sodium chloride, 2.0 g magnesium sulfate, 50 mL 1 M Tris-CI pH 7.5, and 5 mL 2% gelatin per liter). The phage were replated by mixing 0.001 L of phage stock with 100 L
C600 cells in 10 mm, magnesium sulfate, incubating at 37 C for 15 minutes, adding 3 mL LB + 0.7% agarose and plating onto 82 mm LB plates. After ovemight incubation at 37 C followed by 1 hour at 4 C, the phage plaques were transferred to nitrocellulose, and reprobed as above to labeled oligonucleotide probe 2A.
Following the tertiary hybridization screen, 16 phage plaques were isolated.
The inserts of each of the 16 recombinant phage were amplified by PCR using the commercially available lambda gt10 amplimers (Clontech) and the GeneAmp Kit (Perkin-Elmer, Cetus Industries, Norwalk, CT) following the manufacturer's protocols exactly. The amplified DNA was subjected to electrophoresis through 1.2% agarose gels in Tris-EDTA-Acetate (TEA = 40 mM
Tris-Acetate, 1 mM EDTA) buffer, for 2-3 hours at 100 volts. The DC21 a agarose gels were then stained in ethidium bromide (EtBr), rinsed in water and photographed. The DNA was then transferred from the gel to a nitrocellulose membrane by the method of Southem.
A Southem hybridization was performed using labeled oligonucleotide probe 2A in 50 mL hybridizaiton buffer (above) at 40 C overnight then washing at 45 C, 48 C and 51 C. Two amplitied inserts, corresponding to phage no. 64 and no. 76 (Figure 1), hybridized to probe 2A at 51 C in 2 X SSC. Lambda DNA of these 2 clones was prepared following the plate lysale procedure (Sambrook, gta(,, 8upra, p. 2.118). One-tenth (5 mL of 50m1) of the phage DNA was digested with 20 units of the restriction enzyme Eco RI (New England Biolabs, Beverly, MA) in the manufacturer's buffer at 37 C for 2 hours and subjected to agarose gel electrophoresis. Upon EcoRl cleavage of these phage, no. 64 yielded a 1.0 kb insert fragment and the cDNA from phage no. 76 yielded two EcoRl pieces, of 0.9 kb and 0.4 kb.
These bands were excised from the gel.
DNA was eluted from the agarose gel slices by first forcing the gel slices through a 21 gauge needle into 3 mL of T, oEyN,3(10 mM Tris-CI pH 7.4, 1 mM EDTA pH 8.0 and 0.3 M sodium chloride) and freezing at -20 C overnight. The samples were then thawed at 37 C for 30 minutes, centrifuged to pellet the agarose, diluted 1:1 with water and passed through an Elu.Tip column (Schleicher & Schuell) following the manufacture's protocol. The DNA
samples were then ethanol precipitated, ethanol washed, and resuspended to an approximate concentration of 0.05 pmoles/ L.
The plasmid vector bluescript SK+ (Stratagene) was prepared to receive the cDNA inserts by digestion with 20 units of the restriction endonuclease Eco RI (New England Biolabs) in the manufacturer's buffer at 37 C for 2 hours, followed by a 30 minute treatment with 1 unit of calf alkaline phosphatase (Boehringer-Mannheim) which is added directly to the Eco RI reaction. This DNA was then electrophoresed through a 1.2% agarose/TEA gel 209110?

DC21a at 100 volts for 2 hours. The linear plasmid band was excised, eluted and resuspended as above.
cDNA insert fragments were ligated into the prepared bluescript plasmid vector by mixing 0.05 pmole of vector with 0.10 pmoles of cDNA insert in 50 mM Tris-CI pH 7.4, 10 mM
magnesium chloride, 1 mM DTT, 1 mM ATP, and 40 units T4 DNA
ligase (New England Biolabs). The 10 L reaction was incubated at 15 C ovemight. The ligation reaction was then mixed with 100 L of transformation competent E. coli cells, strain DH5a (Bethesda Research Laboratories), and the plasmid DNA
transformed into the E coli cells following the standard protocol of Sambrook et al., supra, p. 1.74. Transformed cells were plated on LB-agar plates containing 100 g/mL ampicillin and grown overnight at 37 C.
Plasmid DNA was isolated from ampicillin resistant colonies following the alkaline lysis procedure of Birnboin and Doly [Nucleic Acids Res. Z, 1513-23 (1979)]. The purified plasmid DNA
was digested with Eco RI as above, subjected to agarose gel electrophoresis and analyzed for the generation of the correct size Eco Ri cDNA insert fragment. Cells from a unique colony positive for a cDNA insert were used to innoculate 100 mL of LB containing 100 g/mL ampicillin and grown to saturation at 37 C. Plasmid DNA was extracted using a Qiagen plasmid isolation kit no.
12143(Qiagen, Inc., Chatsworth, CA) following the manufacturer's protocol.
Sequencing of cDNA clones was performed with the Applied Biosystems, Inc. (ABI, Foster City, CA) 373 Automated DNA Sequencer utilizing either dye-labeled primers or dye-labeled dideoxynucleotides. Cycle sequencing with dye-labeled primers was performed with Taq Dye Primer Cycle Sequencing Kits (ABI part nos. 401121 and 401122). One g of double-stranded DNA was used per reaction. Methods used for cycling and concentration of sequencing samples were as described in the Cycle Sequencing of DNA with Dye Primers manual (ABI part DC21 a no. 901482). Alternatively, cycle sequencing with dye-labeled dideoxynucleotides was performed using the Taq Dye- Deoxy' Terminator Cycle Sequencing Kit (ABI part no. 401113). Typically, 1.25 g of template with 4 pmol of primer was used per reaction.
The template and primer concentrations were varied as necessary to optimize sequencing reactions. Cycling of reactions was performed using a Perkin-Elmer Cetus thermal cycler (model 9810) as described in the Taq Dye Deoxr Terminator Cycle Sequencing Protocol (ABI part no. 901497).
Following the cycle reactions, Centri-Sep"'' spin columns (Princeton Separations, Adelphia, NJ) were used to remove excess dye terminators and primers. Spin column eluants were then precipitated and washed as described in the Taq Dye Deoxy`"' Terminator Cycle Sequencing Protocol (ABI part no.
901497). A 6% acrylamide denaturing gel was prepared as described in the ABI 373A DNA Sequencing System User's Manual. Just prior to running the gel, samples were resuspended in 5 L of deionized formamide/50 mM EDTA (pH 8.0) 5/1 (v/v).
Samples were denatured at 90 C for two minutes, cooled quickly on ice, then loaded onto a pre-run gbl (gel was prerun for approximately 15-20 minutes). The gel was run for 12 hours at the following settings: 2500 vofts, 40 amps, 30 watts, 40 C.
Sequence analysis was performed with ABI 373A DNA Analysis software (version 1Ø2). Final sequence was obtained using ABI
DNA Sequence Editor software seqEe (version 1.0) ABI, Inc..
The entire 1036 bp insert of clone no. 64 was sequenced. It encoded 936 bp of open reading frame continuing through the 3 prime end of the insert (corresponding to a polypeptide with a molecular weight of at least 34,000). Comparison of the sequence of this clone to available sequence in nucleotide sequence data banks revealed that the first 91 bases at the 5' end of the clone corresponded to the bovine mitochondrial genome. Therefore, the 1036 bp insert of clone no. 64 resufted from the ligation of two independent cDNAs during the construction of this library.

209110?
DC21 a The 400 bp EcoRl fragment of clone no. 76 was sequenced entirely indicating 81 bp of open reading frame followed by 298 bases of 3 prime untranslated sequence and a poly A region.
The lambda gt10 bovine small intestine cDNA library was rescreened with an oligonucleotide probe 37A, an exact 33 bp match to the 5' most peptide sequence encoded by clone no. 64.
Two positive clones, no. 22 and no. 23 (Figure 1) were isolated through tertiary screens, subcloned and sequenced as for clone no. 64.
Clones no. 22 and 23 contained 2.8 kb and 1.7 kb cDNA
inserts respectively. The 2.8 kb cDNA insert of clone no. 22 predicted a continuous open reading frame of 835 amino acids between bases 2 and 2506 (corresponding to a 93.2 kDa polypeptide), followed by 298 base of 3' untranslated sequences and a poly A region.
The lambda gt10 library was rescreened with probe 19A, an exact match to the sequence of clone no. 22 corresponding to the 5'-most peptide encoded by that clone, and clone no. 2 was isolated as above. DNA sequence analysis of the 1 kb cDNA
insert from clone no. 2 indicated it overlapped clone no. 22 and extended the 5' end of the bovine cDNA by 100 bases. A
composite of the DNA sequences of clones no. 2 and no. 22 and the predicted translation product is shown in SEQ. ID. NOS. 1 and 3, respectively.
In summary, sequencing of bovine small intestine cDNA
clones corresponding to the 88 kDa component of MTP yielded 2900 bp of continuous sequence which encodes an open reading frame of 860 amino acids followed by a 298 bp 3' noncoding region and a poly A region. The predicted protein product of this composite sequence is 96.1 kDa.

DC21 a Examlile 2 DNA Hybridization Analysis of Related Species Southem hybridization analysis was performed on DNAs from cow, human, mouse, hamster (Chinese hamster ovary or CHO cells), rat, and dog. 10 g of each genomic DNA (Clontech) was digested with 140 units of Eco RI (New England Biolabs) in 100 L 1 X Eco RI buffer (New England Biolabs) at 37 C, overnight. Each digestion reaction was spun at 2,000 rpm in a Uftrafree-MC 10,000 NMWL 1'ilter unit (no. UFC3 TGC 00 from Millipore) with a molecular weight cut-off of 10,000, for 30 minutes to reduce the reaction volume to 50 L. The restriction digest reactions were then subjected to agarose gel electrophoresis through a 0.75% gel in TEA buffer at 80 voRs for 3 hours. The agarose gel was stained with ethidium bromide, photographed, and then transferred to a nitroceliuiose membrane by the method of Southern.
A Southern hybridization was performed using the 2.4 kb Eco RI fragment from the bovine cDNA clone no. 22 as a probe.
Twenty-five ng of the DNA fragment was labeled using the Multiprime DNA Labelling System (Amersham Corp., Arlington Heights, IL) and 50 Ci of 32P-a-dCTP. Unincorporated 32p was separated from the labeled probe using a Sephadex G25 spin column as above. The nitroceilulose membranes was prehybridized in 100 mL hybridization buffer (above) at 37 C for 2 hours. The hybridization was performed ovemight in 50 mL fresh hybridization buffer at 60 C with 1.2 X 107 dpm denatured probe.
The membrane was washed in 500 mL 1 X SSC, 0.1 % SDS at 65 C for 1 hour, air-dried, and then exposed to X-ray film at -80 C
with an intensifying screen for 4 days. The 2.4 kb Eco RI fragment from bovine clone no. 22 specifically hybridized to at least two DNA bands in every species tested. Therefore, it was concluded that the hybridization conditions established for the bovine cDNA
probe allows detection of homologous DNAs from other species, such as human, mouse, hamster, rat and dog.

DC21 a xa ie 3 -isoiation and DNA Sequence Analysis of cDNA
Clones encoding the 88 kDa Component of Human MTP
A. Cloning and Sequence Analysis To obtain the full coding sequence of the 88 kDa component of human MTP, a human liver cDNA library was screened with a bovine MTP cDNA insert described herein above.
The library was obtained from Stratagene. It contained oligo dT
primed liver cDNA directionally cloned (EcoRl to Xhol) into the lambda ZAP vector. The probe was obtained by digestion of 10 g of bovine intestinal clone no. 22 above in universal buffer (Stratagene) with 50 units of EcoRl, electrophoresis at 80-150 voits through a gel consisting of 0.9% low meRing point agarose (Bethesda Research Laboratories, Gaithersburg, MD), TAE (40 mM Tris acetate, 1 mM EDTA), and 0.5 g/mL ethidium bromide.
The resulting 2.4 Kb fragment was purified by phenol extraction as described in Sambrook gLAL, supra, p. 6.30. The purified fragment was then radiolabelled with a muRiprime DNA labelling kit and alpha 32P dCTP (Amersham) to 109 cpm/ g using the manufacturer's instructions. Unincorporated 32p was separated from the labeled probe using a Sephadex G-25 spin column as above.
106 plaques from the library were screened as follows according to the manufacturer's instructions (Stratagene). E. coli bacteria, strain XL I Blue (Stratagene), were grown with shaking overnight at 37 C in 50 mL LB broth (Bethesda Research Laboratories) supplemented with 0.2% maltose and 10 mM
magnesium sulfate. The cells were sedimented by low speed centrifugation and then resuspended in 10 mm, magnesium sulfate to an ODso= 0.5 and stored at 4 C. Phage were diluted to a concentration of 50,000 plaque forming units/25 L SM buffer. For each plate, 600 L of bacteria, and 25 L of phage were mixed DC21 a and incubated at 37 C for 15 minutes. Top agar (6.5 mL) consisting of NZY broth (Bethesda Research Laboratories), 0.7%
agarose (Bethesda Research Laboratories) preheated to 50 C, was added to the bacteria and phage mixture, and then plated onto a 150 mm NZY plate. The top agar was allowed to solidify and the plates were incubated overnight at 37 C.
The plates were then cooled to 4 C for 2 hours and the plaques were lifted onto nitrocellulose filters. Duplicate lifts were performed in which the alignment of the membranes relative to the plate were recorded by placing needle holes through the fi(ter into the agar plate. The filters were incubated 1 minute in 0.5 ,d, sodium hydroxide, 1.5 M sodium chloride, 1 minute in 1 M Tris, pH
8.0, 3 M sodium chloride, and 1 minute in 2 x SSC. Fiiters,were then baked at 80 C in a vacuum chamber for 2 hours. The filters were incubated for 2 hours at 60 C in 5 mL per fiiter of hybridization buffer (6 X SSC, 20 mM NaPO4, 2 X Dendardts, 0.1% SDS, and 100 g/mL salmon sperm DNA). The buffer was replaced with an equal volume of hybridization buffer containing the probe at a concentration of 3.5 x 106 cpm per fifter and incubated overnight at 60 C. The filters were washed in 1 X SSC, 0.1% SDS first at room temperature and then at 50 C for 2 hours.
Autoradiography revealed 56 positives.
A small plug of agarose containing each positive was incubated overnight at 4 C with 1 mL of SM buffer and a drop of chloroform. The positive phage were purified by replating at a!ow density (approximately 50 - 500 per 100 mm plate), screening and isolating single positive plaques as described above.
When XL1 Blue cells are infected with the ZAP vector (Stratagene) and coinfected with a helper phage, the bluescript part of the vector is selectively replicated, circularized and packaged into a single stranded phagemid. This phagemid is converted to a double stranded plasmid upon subsEquent infection into naive XL1 Blue cells. The cDNA insert of the resultant plasmid can be sequenced directly. Plasmids containing DC21 a the positive human liver cDNA inserts were excised in this manner utilizing the helper phage provided by Stratagene according to the manufacturer's directions.
DNA from these clones was purified as follows. A single colony was inoculated into 2 mL of LB and incubated with shaking at 37 C ovemight. 1.5 mL of this was centrifuged and resuspended in 50 L of LB. 300 L of TENS (1 X TE, 0.1 N
sodium hydroxide, 0.5% SDS) was added and vortexed for 5 seconds. 150 L of 3M sodium acetate, pH 5.2 was added and vortexed for 5 seconds. The samples were then spun in a microfuge for 10 minutes. The supernatant was recovered, 0.9 mL
of ethanol was added and the samples were spun in a microfuge for 10 minutes. The pellet was washed in 70% ethanol, dried, and resuspended in 20 L of TE (10 mM Tris pH 7.4, 1 mM EDTA pH
8).
The DNA from the clones was characterized as follows.
Five L of the DNA from each clone were digested with 10 units Eco RI, 10 units Xhol, and 10 g RNAse, and then fractionated and visualized by electrophoresis through a 1% agarose, TBE (45 mM Tris-Borate, 1 mM EDTA), 0.5 g/mL ethidium bromide gel. A
Southern blot of the gel was performed as described in Sambrook et ai., supra, p. 9.41. This Southern blot was probed with a fragment of the bovine cDNA near the 5' end of the coding sequence. This 5' probe was prepared by digesting 25 g of bovine intestinal clone no. 2 above with 50 units EcoRl and 50 units of Nhel, isolating as above the 376 base pair fragment from a 2% low melting point agarose, TBE, 0.5 ug/mL ethidium bromide gel, and radiolabelling as described above. The resufts are as follows: Clone no. 693, 3.7 kB insert, hybridizes with the 5' probe;
Clone no. 754, 1.2 kB insert, hybridizes with the 5' probe; Clone no. 681, 1.8 kB insert, does not hybridize with the 5' probe.
Ovemight cultures containing these three clones were grown in 200 mL of LB with 100 g/mL ampicillin. Large amounts of plasmid were purified using a Qiagen plasmid maxiprep kit DC21 a according to the manufacturer's instructions. The sequence of clone no. 693 reveals that it contained two inserts. The 5' 500 bp insert was homologous to haptoglobin and will not be discussed further. This was followed by a mutant Xhol and an EcoRl restriction site (the two sites used in the directional cloning). The 3' insert was the cDNA of interest. It contained some 5' untransiated sequence as indicated by the stop codons in all three reading frames. At bases 48 - 2729 there is an ATG-initiated open reading frame corresponding to 894 amino acids. The deduced amino acid sequence begins M I L L A V L F L C F I
(SEQ. ID. NO. 28). The stop codon is found at bases 2730 - 2732 followed by a 3' untransiated region of 435 bases and a poly A
region. The sequence of clone no. 681 confirmed the 3' 1768 bases of this clone, and clone no. 754 confirmed bases 1 through 442.

B. Tissue Localization of the 88 kDa mRNA
A MultiTissue Northem Blot (Clontech) contained 2 g per lane of poiyA+ RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney or pancreas. Northern hybridization was performed as for the genomic Southern blot. Prehybridization was in 50 mL hybridization buffer at 37 C for 2 hours followed by an overnight hybridization in 20 mL fresh buffer at 60 C with 5.2 x 107 dpm labeled 2.4 kb Eco RI fragment from the bovine intestinal clone no. 22 as above. The Northern blot was washed in 500 mL
0.2 X SSC, 0.1% SDS at 60 C, 1 hour and subjected to autoradiography at -80 C. After a 20 hour exposure to X-ray film there is a predominant signal in the liver RNA lane at about 4.4 kb and no other detectable hybridization. Therefore, cross hybridization of the 2.4 kb fragment of the bovine cDNA detects a human liver RNA specifically. As liver and intestine are the only two tissues in which significant MTP activity has been reported, the cloning and northern blot analysis support the biochemical localization for MTP. Also, the results of the northern analysis DC21 a extend this detection to include DNA:RNA hybrids as well as DNA:DNA interactions.

Exam iR e 4 Expression of MTP In Human Fibroblast Cell Line 1. Methods All standard molecular biology protocols were taken from Sambrook, supra, except where indicated below. All restriction enzymes used in this example were obtained from Bethesda Research Laboratories (BRL, Gaithersburg, MD). A 3.2 kb fragment extending from nucleotide -64 to 3135 (relative to the translation start site with A of the translation start site ATG codon designated +1), was constructed from plasmids p754 (bases -64 to 384) and p693 (bases 385 to 3135) as follows. A 448 bp EcoRl-Ncol restriction endonuclease fragment and a 2750 bp Ncot-Xhol restriction endonuclease fragment were excised from p754 and p693, respectively. Following gel purification, the fragments were ligated into EcoRI-Xhol cut plasmid pBluescript-SK to yield plasmid pBS/hMTP. The entire hMTP fragment was isolated from pBS/hMTP by restriction endonuclease digestion with Hindill and Xhol and was subcloned into plasmid pcDNA/Neo (Invitrogen,San Diego, CA) to yield plasmid pcDNA/MTP. This places the full-length hMTP coding sequence under the transcriptional control of the highly active Cytomegalovirus promoter.
Plasmids were transfected into 1508T [J. Biol. Chem. 2&Z, 13229-38 (1992)] transformed human skin fibroblasts by the lipofectin reagent (BRL). Cells were split into 100 mm dishes at a density of 25% of confluency, 24 hours prior to transfection. At the time of transfection, 50 mg of plasmid per 100 mm plate were dissolved in 1.5 mL of serum-free Dulbecco's Modified Eagles Medium (DMEM) and added dropwise to a solution of 120 L
lipofectin reagent in 1.5 mL of serum free DMEM. After a 15-minute incubation at room temperature, the transfection mixtures were added to the 1508T cultures containing 7 mL of serum free DC21 a DMEM. Twenty four hours later, the transfection mixtures were removed and 10 mL of fresh DMEM containing 10% fetal bovine serum was added for an additional 24 hours. Cells were scraped from the dish and washed twice with ice cold phosphate buffered saline (PBS). Cell extracts, MTP activity measurements and Westem analyses were canied out as described in the foregoing "Assay for TG transfer activity in Abetalipoproteinemic subjects"
herein.

ii. Results The cDNA containing the full coding sequence for MTP was subcloned into expression vector pcDNA/Neo, yielding construct pcDNA/MTP. This plasmid was transiently expressed in 1508T
transformed human skin fibroblasts (J. Biol. Chem. =, 13229-38 (1992)] by liposome mediated transfection. Forty-eight hours after transfection, TG transfer activity was readily detectable above background levels assayed in extracts from cells transfected with the parent plasmid, pcDNA/Neo. Western blot analysis showed the presence of the the 88 kDa component of MTP in cells transfected with pcDNA/MTP but not in cells transfected with pcDNA/Neo. A comparison of the protein mass and activity in the transfected cells to that found in HepG2 cells suggests that the expressed MTP was efficiently incoporated into an active transfer protein complex with PDI.
Example 5 Screen for Identifying Inhibitors of MTP
In this screen, the rate of detectably labeled lipid (for example, NMR, ESR, radio or fluorescently labeled TG, CE, or PC) transfer from donor particies (e.g., donor membranes, vesicles, or lipoproteins) to acceptor particles (e.g., acceptor membranes, vesicles, or lipoproteins) in the presence of MTP is measured. A
decrease in the observed transfer rate in the presence of an inhibitor of MTP (e.g., contained in a natural products extract or DC21 a known compounds) may be used as an assay to identify and isolate inhibitors of MTP function. A variety of assays could be used for this purpose, for example, the synthetic vesicle assays previously published by Wetterau & Zilversmit, J. Biol. Chem. ZU, 10863-6 (1984) or Wetterau et al.. J. Biol. Chem. M, 9800-7 (1990) or the assay outlined hereinabove in the "Assay for TG
transfer activity in Abetalipoproteinemic subjects." An example of one such assay is as follows.
A. Substrate Preparation In a typical screen using labeled lipoproteins, labeling of lipoproteins with [3H]-TG is accomplished by the lipid dispersion procedure described by Morton and Zilversmit [Morton, R.E. ILaL, J. Biol. Chem. L%, 1992-5 (1981)] using commercially available materials. In this preparation, 375 Ci of [3H] triolein (Triolein, [9,10-3H (N)]-, NEN Research Products, cat. no. NET-431), 1.5 mg of egg phosphatidylcholine and 160 g of unlabeled triolein in chloroform are mixed and evaporated under a stream of nitrogen to complete dryness. Two mL of 50 mM Tris-HCI, 0.01%
Na2 EDTA, 1 mM dithiothreitol, pH 7.4, is added and the tube flushed with nitrogen. The lipids are resuspended by vortexing and the suspension is then sonicated for two 20-minutes intervals in a bath sonicator. The sonicated lipids are added to 75 mL
rabbit plasma (Pei-Freez Biologicals, Rogers, AR) with 5.8 mL of 8.2 mM diethyl R nitrophenyl phophate (Sigma, Cat. No. D9286) and 0.5 mL of 0.4 M Na2EDTA, 4% NaN3. The plasma is then incubated under nitrogen for 16-24 hours at 37 C. Low density lipoproteins (LDL) and high density lipoproteins (HDL) are isolated from the incubation mixture and from control plasma which was not labeled by sequential ultracentrifugation [Schumaker & Puppion, Methods Enzymology ]M, 155-170 (1986)]. Isolated lipoproteins are dialyzed at 4 C agairist 0.9%
sodium chloride, 0.01% Na2EDTA, and 0.02% NaN3 and stored at 4 C.

DC21 a B. Transfer Assay In a typical 150 L assay, transfer activity is determined by measuring the transfer of radiolabeled TG from [3H]-HDL (5 ug cholesterol) donor particles to LDL (50 g cholesterol) acceptor particles at 37 C for three hours in 15 mM Tris, pH 7.4, 125 m,pd MOPS, 30 mM Na acetate, 160 mM NaCI, 2.5 mM Na2 EDTA, 0.02% NaN3, 0.5% BSA with about 50-200 ng purified MTP in the well of a 96-well plate. The materiai to be tested (e.g., natural product extracts in an assay compatible solvent such as ethanol, methanol or DMSO (typically, 5 L of material in 10% DMSO is added) can be screened by addition to a well prior to incubation.
The transfer is terminated with the addition of 10 L of freshly prepared, 4 C 1leparin/MnC12 solution (1.0 g heparin, Sigma Cat.
No. H3393 187 U/mg, to 13.9 mL, 1.5 m, MnC12. 0.4% heparin (187 I.U.)/0.1 M MnC12) to precipitate the 3H-TG-LDL acceptor particles and the plate centrifuged at 800 x g. An aliquot of the supernatant from each well containing the [sH]-TG-HDL donor particles is transferred to scintillation cocktail and the radioactivity quantitated. The enzyme activity is based on the percentage of TG
transfer and is calculated by the following equation:
[3H]-TG recovery (+ MTP) Enzyme activity = 1 - x 100%
[3H]-TG recovery (- MTP) In such an assay, the percent TG transfer will increase with increasing MTP concentration. An inhibitor candidate will decrease the percent TG transfer. A similar assay could be performed with labeled CE or PC.

xamwg 6 identification and Demonstration of the Activity of MTP inhibitors DC21 a i. Methods A. Identification of MTP Inhibitors -Using the method outlined in Example 5, MTP inhibitor compounds A and B were identified. The assay measured the bovine MTP-catalyzed rate of transport of radiolabeled TG from donor HDL to acceptor LDL. In this method, an inhibitor decreases the rate of radiolabeled TG transfer.
The MTP-inhibiting activity of these compounds was confirmed in an independent assay following the procedures outlined in the foregoing "Assay for TG transfer activity in abetalipoproteinemic subjects." That assay measured the bovine MTP-catalyzed transport of radiolabeled TG from donor to acceptor SUV.
B. Ceii culture The human hepatoblastoma cell line, HepG2, was obtained from the American Type Culture Collection (Rockville, MD; ATCC
accession no. 8065). Cultures were maintained at 37 C in a 5%
carbon dioxide atmosphere in T-75 cufture flasks with 12 mL of RPMI 1640 medium containing 10% fetal bovine serum (all cell culture media and buffers were obtained from GIBCO Life Technologies, Gaithersburg, MD). Cells were subcuitured 1:4 once a week and fed fresh mediurri 3 times a week.
Experiments to measure the effects of compounds A and B
on protein secretion were carried out in 48-well plates. HepG2 cells were subcuRured 1:2 and allowed to come to confluency at least 24 hours before drug treatment. Before commencement of drug treatment, culture medium was removed, the cells washed once with PBS and 1 mL of fresh medium was added quantitatively. Compound A was added to duplicate wells in 10 L
of dimethylsulfoxide (DMSO) to yield varying compound concentrations. DMSO alone (10 L) was used as the negative control. (Note: DMSO at this concentration has negligible effect on HepG2 cells.) After a 16-hour incubation under standard cell culture conditions, the plates were centrifuged at 2,500 rpm for 5 minutes at 4 C to sediment any loose cells. The media were diluted with cell cutture medium 10 times for the apolipoprotein B
(apoB) and human serum albumin (HSA) assays, and 20 times for the apolipoprotein Al (apoAl) assays. The cells were washed twice with cold PBS, and 0.5 mL of homogenization buffer was then added (0.1 M sodium phosphate, pH 8.0; 0.1 % Triton X-100).
The cells were homogenized by trituration with a 1 mL
micropipettor, and protein was measured using the Coomassie reagent (Pierce Chemical Co, Rockford, IL) as described by the manufacturer.
C. ELISA assays for ApoB and ApoAl and HSA
The ELISA assays to measure protein mass were of the "sandwich" design. Microtiter plates were coated with a monoclonal antibody (primary antibody), specific. for the protein of interest (Biodesigns International, Kennebunkport, ME), followed by the antigen or sample, a polyclonal antibody (secondary antibody) directed to the protein of interest (Biodesigns International), and a third antibody conjugated to alkaline phosphatase directed to the secondary antibody (Sigma Biochemical, St.Louis, MO). The 96-well microtiter plates (Coming no. 25801) were coated ovemight at room temperature with 100 L of diluted monoclonal antibody (final concentrations were 1 g/mL, 2 g/mL and 4 g/mL for anti- apoB, apoAl and HSA, respectively, in 0.1 M sodium carbonate-sodium bicarbonate, pH
9.6 and 0.2 mg/mL sodium azide). Coating was carried out overnight at room temperature. After coating and between each subsequent incubation step, the plates were washed five times with 0.9% sodium chloride with 0.05% TweenTM 20. Duplicate aliquots (100 L) of diluted culture media or standard (purified apoB, apoAl or HSA diluted to 0.3125-320 ng/mL with cell culture medium) were added to wells coated with monoclonal antibody.
Following incubation for 1.5 hours at room temperature, the antigen or sample was removed and the wells washed. The secondary antibodies were diluted 1:500 in PBS + 0.05% TweenTM

DC21a 20 (Buffer III), then 100 L was added to each well and incubated for 1 hour at room temperature. The antibody was rerfioved and the wells were washed. All secondary antibodies were polyclonal antisera raised in goat against the human proteins. A rabbil anti-goat IgG, conjugated to alkaline phosphatase, was diluted 1:1000 with Buffer III and 100 L was added to each well. Following incubation for 1 hour at room temperature, the antibody was removed and wells washed eight times. The substrate p-nitrophenylphosphate (Sigma Biochemical, St. Louis, MO) was added at 1 mg/mL in 0.05 M NaCarbonate-NaBicarbonate, pH 9.8 + 1 mA& magnesium chloride. Following a 45-minute reaction at room temperature, the assay was stopped and the color stabilized with the addition of 100 L of 0.1 M Tris, pH 8.0 + 0.1 M EDTA.
The microtiter plates were read at 405 nm in a V-Max 96-well plate reader (Molecular Devices, Menlo Park, CA).
After subtraction of background, the standards were plotted on a semi-log graph and logarithmic regression was performed.
The equation for the curve was used to calculate the concentration of apoB, apoAl and HSA. The protein concentration was normalized to total cell protein yielding concentrations with units of ng/mVmg cell protein. Each drug treatment was performed in duplicate and the resutts were averaged. The apoB, apoAl, and HSA concentrations for each drug treatment were divided by the corresponding protein concentration in the DMSO control. The results were plotted as a percentage of control versus the drug concentration.
D. Lipid analysis HepG2 cells were subcuftured into 6-well dishes and allowed to come to confluency at least 24 hours before drug treatment. Prior to addition of the drug, culture media were removed, cells washed once with PBS, and 1 mL of fresh medium (RPMI 1640 + 10% FBS) was added quatitatively. Compound A
was added to duplicate wells in 10 L of DMSO to yield varying compound concentrations. DMSO alone (10 L) was used as the DC21 a negative control. After a 16-hour incubation under standard cell cufture conditions, the media were removed and 1 mL-of labeling medium (RPMI 1640; 16.5 mg/mL fatty acid free BSA; 1 mM
sodium oleate; 1 mm, glycerol; 5 Ci/mL 3H-glycerol (Amersham, Arlington Heights, IL, Catalog no. TRA.244) was added with a second addition of compound A. The cultures were incubated for 2 hours under standard cell culture conditions. Media (1 mL) were removed to 15-mL glass tubes and immediately diluted with 2 mL
of ice cold methanol and 1 mL, of dH2O. Cells were washed once with PBS and were processed for total protein measurements as described in section I-B.
Total lipids were extracted from the media and analyzed as follows. After addition of 5.0 mL of chloroform and 0.2 mL of 2%
acetic acid, the tubes were vortexed for 1 minute and centrifuged at 2,000 rpm for 5 minutes to separate the aqueous and organic phases. The upper aqueous phase was removed and 3.6 mL of methanol:water (1:1) containing 0.1% acetic acid added. After briefly vortexing, the tubes were centrifuged as before and the aqueous phase again removed. The organic phase was quatitatively transferred to clean 15-mL glass tubes and the solvent evaporated under nitrogen. Dried lipids were dissolved in 0.1 mL of chloroform and 30 L of each sample were spotted onto silica gel 60A, 19 channel thin layer chromatography plates (Whatman). 5-10 g of TG in 10 L of chloroform were added as carrier and the plates were developed in hexane:diisopropyl ether:
acetic acid (130:70:4, VN). After drying, lipid was stained by exposing the plates to iodine. Bands corresponding to TG were scraped into scintillation vials. 0.5 mL of dH2O and 10 mL of EcoLite (ICN Biomedical) scintillation fluid were added and the samples vortexed vigorously. Raw data was normalized to cell protein and expressed as percent of DMSO control.

DC21 a II. Results A. Identification of MTP inhibitors -The primary screen suggested that compound A inhibited the MTP-catalyzed transport of 3H-TG from HDL to LDL. The ability of compound A to inhibit MTP-catalyzed lipid transport was confirmed in a second assay which measures the MTP-catalyzed transport of 3H-TG from donor SUV to acceptor SUV. The IC50 for compound A is about 1 M (Figure 10).
B. Inhibition of apoB and TG secretion Compound A was administered to HepG2 cells in a twofold dilution series ranging from 0.156 to 20 M. After a 16-hour incubation under standard cell culture conditions, aliquots of the conditioned media were assayed by ELISA for apoB, apoAl and HSA. ApoB secretion was inhibited in a dose-responsive manner with an IC50 of 5 gM (Figure 11). The secretion of apoAl and HSA
was unaffected up to the maximum dose of 20 M confirming that the inhibition was specific for apoB. These data indicate that addition of an MTP inhibftor to a human liver cell line inhibits the secretion of lipoproteins which contain apoB.
HepG2 cells were treated with doses of compound A
ranging from 1.25 ,pd - 20 M under conditions identical to those utilized for the apoB, apoAl and HSA secretion experiment. The intracellular pool of TG was radiolabelled for two hours with 3H-glycerol in the presence of vehicle or varying doses of compound A. The accumulation of radiolabelled TG in the medium was measured by quantitative extraction, followed by thin layer chromatography analysis and normalization to total cell protein.
DMSO alone was used as a control. TG secretion was inhibited by compound A In a dose-dependent manner. The ICsp was observed to be about 2.0 M, which is similar to the IC50 for inhibition of apoB secretion (Figure 12). The data confirm that compound A inhibits the secretion of TG-rich lipoproteins that contain apoB.

DC21 a The foregoing procedures were repeated with compound B.
Compound B inhibits MTP-catalyzed 3H-TG transport from donor SUV to acceptor SUV. The IC50 is about 4 to 6 M (Figure 13).
The secretion of lipoproteins that contain apoB is also inhibited in HepG2 ceils by compound B (Figure 141.
Example 7 Inhibition of MTP-cataiyzed CE and PC Transport 1. Methods To measure the effect of compound A on bovine MTP-catalyzed transport of CE or PC between membranes, the lipid transfer assay which measures TG transfer between SUV was modified. The composition of the donor vesicles was the same, except 0.25 mol% 14C-CE or 14C-PC replaced the labeled TG.
The composition of the acceptor vesicles were the same, except labeled PC and unlabeled TG were not included. Following precipitation of donor vesicles, the percentage of lipid transfer was calculated by comparing the 14C-CE or -PC in the acceptor vesicles in the supernatant following a transfer reaction to the total 14C-CE or -PC in the assay. The labeled lipid in the supernatant in the absence of MTP was subtracted from the labeled iipid in the presence MTP to calculate the MTP-catalyzed lipid transfer from donor SUV to acceptor SUV. The remainder of the assay was essentially as described previously.
11. Results The ability of compound A to inhibit the MTP-catalyzed transport of radiolabeled CE and PC between membranes was also investigated. Compound A inhibited CE transfer in a manner comparable to its inhibition of TG transfer. Compound A Inhibited PC transfer, but it was less effective at inhibiting PC transfer than CE and TG transfer. Approximately 40% of the PC transfer was inhibited at concentrations of inhibitor which decreased TG and CE transfer more than 80%.

DC21 a Example 8 Cloning of bovine MTP - 5' end A bovine small intestinal cDNA library, packaged in lambda gt10, was obtained from Clontech (#BL1- 010A). The library was diluted in SM to contain 50,000 phage/100 L (a 1:100,000 dilution). The diluted phage (100 L) were mixed with 300 L
E.Coli C600 cells (Clontech) and incubated for 15 minutes at 37 C. After adding 7 mL of top agarose, the mixture was poured onto a 150 mm plate containing 75 mL of LB agarose. A total of 25 plates, each containing approximately 5 X 104 phage, were prepared in this manner. The plates were incubated overnight at 37 C.
To isolate phage DNA, 10 mL SM (no gelatin) was added to each plate. The plates were then rocked gently at room temperature for 2 hours. The eluted phage (approximately 8 mUplate) were collected and pooled. E.Coli cells were sedimented by centrifugation for 10 minutes at 12,000 X g.
Lambda DNA was isolated from the supernatant using the QIAGEN tip-100 (midi) preparation according to the protocol supplied by the manufacturer. The purified DNA was resuspended in a total of 200 L TE (10 mM Tris.Cl pH 8.0, 1 mM EDTA).
1 g lambda phage DNA (approximately 3 X 107 molecules) was added to a 100 L PCR reaction containing 2 mbA
magnesium chloride, 0.2 mM each deoxynucleotide triphosphate, 1.25X buffer, and 2.5 units Taq polymerase (Perkin-Elmer Cetus, kit #N801-0555). The concentration of each primer was 0.15 mIA.
The sequence of the forward primer (SEQ. ID. NO. 29) was as follows:

GGTCAAT$MATTCTTCTTGCTGTGC.
The forward primer's sequence was based on the human cDNA
sequence encoding bases 41 to 66 of the 88 kDa component of MTP. The reverse primer (SEQ. ID. NO. 30) had the following sequence:

DC21 a 658 636 (bovine) 807 785 (human) -GCCTCGATACTATTTTGCCTGCT

The reverse primer's sequence was based on the known bovine cDNA sequence encoding the 88 kDa component of MTP and hybridizes from base 658 to 636 of the bovin3 cDNA, which correspond to bases 807-785 of the human cDNA.
PCR-amplification was conducted in a Perkin-Elmer thermal cycler, model 9600. After a two-minute incubation at 97 C, the reaction was cycled at 94 C for 30 seconds, 50 C for 30 seconds, and 72 C for one minute for 35 cycles. A final incubation at 72 C for 7 minutes was performed.
The PCR product was electrophoresed on a 1% agarose gel in TAE buffer as described previously. The yield of the desired 766 base pair fragment was approximately 2 g. The DNA was excised from the gel, purified using GeneClean (Bio101 La Jolla, CA), blunt-ended, cloned into pUC 18-Sma1 (Pharmacia), and sequenced as described previously.
The new sequence obtained from the bovine cDNA
encoding thn 5' region of the 88 kDa component of MTP is shown in SEQ. ID. NO. 5. The sequence adds 83 bases to the 5' end of the bovine cDNA reported previously.

Example 9 Sequencing of human genomic DNA for the 88 kDa component of MTP
Sequencing of human genomic DNA was carried out by the procedures described in "Demonstration of a gene defect in a second abetalipoproteinemic subject" and in Example 1. The result of this procedure is the human genomic sequence SEQ. ID.
NO. 8.

SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT:
(A) NAME: John R. Wetterau II
(B) STREET: 190 Rugby Drive (C) CITY: Langhorne, PA
(D) COUNTRY: USA

(i) APPLICANT:
(A) NAME: Daru Young Sharp (B) STREET: 893 Perrineville Road (C) CITY: Perrineville, NJ
(D) COUNTRY: USA
(i) APPLICANT:
(A) NAME: Richard E. Gregg (B) STREET: 7 Linden Lane (C) CITY: Pennington, NJ
(D) COUNTRY: USA

(ii) TITLE OF INVENTION: MICROSOMAL TRIGLYCERIDE TRANSFER
PROTEIN

(iii) NUMBER OF SEQUENCES: 30 (iv) CORRESPONDENCE ADDRESS:
OSLER, HOSKIN & HARCOURT
Suite 1500 50 O'Connor Street Ottawa, Ontario (v) COMPUTER READABLE FORM:
(A) COMPUTER: IBM PC compatible (B) OPERATING SYSTEM: PC-DOS/MS-DOS
(C) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,091,102 (B) FILING DATE: March 5, 1993 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/847,503 (B) FILING DATE: March 6, 1992 (viii) PATENT AGENT INFORMATION
(A) NAME: J. Bradley White (B) REFERENCE NUMBER: PCA10215 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2900 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

aaactcacat actccactga agtttttctc gatcggggca aaggaaacct ccaagacagt 60 gtgggctacc gaatttcatc caatgtggat gtcgctttac tgtggaggag tcctgatggt 120 gatgataacc aactgatcca aattacgatg aaagatgtaa accttgaaaa tgtgaatcaa 180 cagagaggag agaagagcat tttcaaagga aaaaagtcat ctcaaatcat aagaaaggaa 240 aacttggaag caatgcaaag acctgtgctc cttcatctaa ttcatggaaa gatcaaagag 300 ttctactcat atcaaaatga accagcagcc atagaaaatc tcaagagagg cctggctagc 360 ctatttcaga tgcagttaag ctctggaact accaatgagg tagacatctc tggagattgt 420 aaagtgacct accaggctca tcaagacaaa gtgaccaaaa ttaaggcttt ggattcatgc 480 aaaatagaga gggctggatt tacgacccca catcaggtct tgggtgtcac ttcgaaagcc 540 acatctgtca ctacctataa gatagaagac agctttgttg tagctgtgct ctcagaagag 600 atacgtgctt taaggctcaa ttttctacaa tcaatagcag gcaaaatagt atcgaggcag 660 aaactggagc tgaaaaccac ggaagcaagc gtgagactga agccaggaaa gcaggttgca 720 gccatcatta aagcagtcga ttcaaagtac acggccattc ccattgtggg gcaggtcttc 780 cagagcaagt gcaaaggatg cccttctctc tcagagcact ggcagtccat cagaaaacac 840 ctgcagcctg acaacctctc caaggctgag gctgtcagaa gcttcctggc cttcatcaag 900 cacctcagga cggcaaagaa agaagagatc ctccaaattc taaaggcaga aaacaaggaa 960 gtactacccc agctagtgga tgctgtcacc tctgctcaga caccagactc attagacgcc 1020 attttggact ttctggattt caaaagcacc gagagcgtta tcctccagga aaggtttctc 1080 tatgcctgtg catttgcctc acatcctgat gaagaactcc tgagagccct cattagtaag 1140 ttcaaaggtt cttttggaag caatgacatc agagaatctg ttatgatcat catcggggcc 1200 cttgtcagga agttgtgtca gaaccaaggc tgcaaactga aaggagtaat agaagccaaa 1260 aagttaatct tgggaggact tgaaaaagca gagaaaaaag aggacatcgt gatgtacctg 1320 ctggctctga agaacgcccg gcttccagaa ggcatcccgc tccttctgaa gtacacagag 1380 acaggagaag ggcccattag ccaccttgcc gccaccacac tccagagata tgatgtccct 1440 ttcataactg atgaggtaaa gaagactatg aacaggatat accaccagaa tcgtaaaata 1500 catgaaaaaa ctgtgcgtac tactgcagct gccatcattt taaaaaacaa tccatcctac 1560 atggaagtaa aaaacatcct gctctctatt ggggaacttc ccaaagaaat gaataagtac 1620 atgctctcca ttgtccaaga catcctacgt tttgaaacac ctgcaagcaa aatggtccgt 1680 caagttctga aggaaatggt cgctcataat tacgatcgtt tctccaagag tgggtcctcc 1740 tctgcatata ctggctacgt agaacggact tcccattcgg catctactta cagccttgac 1800 attctttact ctggttctgg cattctaagg agaagtaatc tgaacatctt tcagtatatt 1860 gagaaaactc ctcttcatgg tatccaggtg gtcattgaag cccaaggact ggaggcatta 1920 attgcagcca ctcctgatga gggggaagag aaccttgact cctatgctgg cttgtcagct 1980 ctcctctttg atgttcagct cagacctgtc acttttttca acgggtacag tgatttgatg 2040 tccaaaatgc tgtcagcatc tagtgaccct atgagtgtgg tgaaaggact tcttctgcta 2100 atagatcatt cccaggagct tcagctgcaa tctggactta aggccaatat ggatgttcaa 2160 ggtggtctag ctattgatat tacaggtgcc atggagttta gtctatggta tcgtgaatct 2220 aaaacccgag tgaaaaatcg ggtaagtgtg ttaataactg gtggcatcac ggtggactcc 2280 tcttttgtga aagctggctt ggaaattggt gcagaaacag aagcaggctt ggagtttatc 2340 tccacggtgc agttttctca gtacccattt ttagtttgtc tgcagatgga caaggaagat 2400 gttccataca ggcagtttga gacaaaatat gaaaggctgt ccacaggcag aggttacatc 2460 tctcggaaga gaaaagaaag cctaatagga ggatgtgaat tcccgctgca ccaagagaac 2520 tctgacatgt gcaaggtggt gtttgctcct caaccagaga gcagttccag tggttggttt 2580 tgaaactgat gggggctgtt tcattagact tcatctcgcc agaagggata agacgtgaca 2640 tgcctaagta ttgctctctg agagcacagt gtttacatat ttacctgtat ttaagagttt 2700 tgtagaacgt gatgaaaaac ctcacataat taagtttggg cctgaatcat ttgatactac 2760 ctacagggtc attctgagcc actctatgtg ataccttagt agcgttctgt tttcctgcat 2820 ctctctcaaa tcacatttac tactgtgaaa ctagttctgc cctaagaaga aaccattgtt 2880 taaaaaaaaa aaaaaaaaaa 2900 (2) INFORMATION FOR SEQ ID NO:2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3185 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

gtgactccta gctgggcact ggatgcagtt gaggattgct ggtcaatatg attcttcttg 60 ctgtgctttt tctctgcttc atttcctcat attcagcttc tgttaaaggt cacacaactg 120 gtctctcatt aaataatgac cggctgtaca agctcacgta ctccactgaa gttcttcttg 180 atcggggcaa aggaaaactg caagacagcg tgggctaccg catttcctcc aacgtggatg 240 tggccttact atggaggaat cctgatggtg atgatgacca gttgatccaa ataacgatga 300 aggatgtaaa tgttgaaaat gtgaatcagc agagaggaga gaagagcatc ttcaaaggaa 360 aaagcccatc taaaataatg ggaaaggaaa acttggaagc tctgcaaaga cctacgctcc 420 ttcatctaat ccatggaaag gtcaaagagt tctactcata tcaaaatgag gcagtggcca 480 tagaaaatat caagagaggt ctggctagcc tatttcagac acagttaagc tctggaacca 540 ccaatgaggt agatatctct ggaaattgta aagtgaccta ccaggctcat caagacaaag 600 tgatcaaaat taaggccttg gattcatgca aaatagcgag gtctggattt acgaccccaa 660 atcaggtctt gggtgtcagt tcaaaagcta catctgtcac cacctataag atagaagaca 720 gctttgttat agctgtgctt gctgaagaaa cacacaattt tggactgaat ttcctacaaa 780 ccattaaggg gaaaatagta tcagggcaga aattagagct gaagacaacc gaagcaggcc 840 caagattgat gtctggaaag caggctgcag ccataatcaa agcagttgat tcaaagtaca 900 cggccattcc cattgtgggg caggtcttcc agagccactg taaaggatgt ccttctctct 960 cggagctctg gcggtccacc aggaaatacc tgcagcctga caacctttcc aaggctgagg 1020 ctgtcagaaa cttcctggcc ttcattcagc acctcaggac tgcgaagaaa gaagagatcc 1080 ttcaaatact aaagatggaa aataaggaag tattacctca gctggtggat gctgtcacct 1140 ctgctcagac ctcagactca ttagaagcca ttttggactt tttggatttc aaaagtgaca 1200 gcagcattat cctccaggag aggtttctct atgcctgtgg atttgcttct catcccaatg 1260 aagaactcct gagagccctc attagtaagt tcaaaggttc tattggtagc agtgacatca 1320 gagaaactgt tatgatcatc actgggacac ttgtcagaaa gttgtgtcag aatgaaggct 1380 gcaaactcaa agcagtagtg gaagctaaga agttaatcct gggaggactt gaaaaagcag 1440 agaaaaaaga ggacaccagg atgtatctgc tggctttgaa gaatgccctg cttccagaag 1500 gcatcccaag tcttctgaag tatgcagaag caggagaagg gcccatcagc cacctggcta 1560 ccactgctct ccagagatat gatctccctt tcataactga tgaggtgaag aagaccttaa 1620 acagaatata ccaccaaaac cgtaaagttc atgaaaagac tgtgcgcact gctgcagctg 1680 ctatcatttt aaataacaat ccatcctaca tggacgtcaa gaacatcctg ctgtctattg 1740 gggagcttcc ccaagaaatg aataaataca tgctcgccat tgttcaagac atcctacgtt 1800 ttgaaatgcc tgcaagcaaa attgtccgtc gagttctgaa ggaaatggtc gctcacaatt 1860 atgaccgttt ctccaggagt ggatcttctt ctgcctacac tggctacata gaacgtagtc 1920 cccgttcggc atctacttac agcctagaca ttctctactc gggttctggc attctaagga 1980 gaagtaacct gaacatcttt cagtacattg ggaaggctgg tcttcacggt agccaggtgg 2040 ttattgaagc ccaaggactg gaagccttaa tcgcagccac ccctgacgag ggggaggaga 2100 accttgactc ctatgctggt atgtcagcca tcctctttga tgttcagctc agacctgtca 2160 cctttttcaa cggatacagt gatttgatgt ccaaaatgct gtcagcatct ggcgacccta 2220 tcagtgtggt gaaaggactt attctgctaa tagatcattc tcaggaactt cagttacaat 2280 ctggactaaa agccaatata gaggtccagg gtggtctagc tattgatatt tcaggtgcaa 2340 tggagtttag cttgtggtat cgtgagtcta aaacccgagt gaaaaatagg gtgactgtgg 2400 taataaccac tgacatcaca gtggactcct cttttgtgaa agctggcctg gaaaccagta 2460 cagaaacaga agcaggcttg gagtttatct ccacagtgca gttttctcag tacccattct 2520 tagtttgcat gcagatggac aaggatgaag ctccattcag gcaatttgag aaaaagtacg 2580 aaaggctgtc cacaggcaga ggttatgtct ctcagaaaag aaaagaaagc gtattagcag 2640 gatgtgaatt cccgctccat caagagaact cagagatgtg caaagtggtg tttgcccctc 2700 agccggatag tacttccagc ggatggtttt gaaactgacc tgtgatattt tacttgaatt 2760 tgtctccccg aaagggacac aatgtggcat gactaagtac ttgctctctg agagcacagc 2820 gtttacatat ttacctgtat ttaagatttt tgtaaaaagc tacaaaaaac tgcagtttga 2880 tcaaatttgg gtatatgcag tatgctaccc acagcgtcat tttgaatcat catgtgacgc 2940 tttcaacaac gttcttagtt tacttatacc tctctcaaat ctcatttggt acagtcagaa 3000 tagttattct ctaagaggaa actagtgttt gttaaaaaca aaaataaaaa caaaaccaca 3060 caaggagaac ccaattttgt ttcaacaatt tttgatcaat gtatatgaag ctcttgatag 3120 gacttcctta agcatgacgg gaaaaccaaa cacgttccct aatcaggaaa aaaaaaaaaa 3180 aaaaa 3185 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 860 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Lys Leu Thr Tyr Ser Thr Glu Val Phe Leu Asp Arg Gly Lys Gly Asn Leu Gln Asp Ser Val Gly Tyr Arg Ile Ser Ser Asn Val Asp Val Ala Leu Leu Trp Arg Ser Pro Asp Gly Asp Asp Asn Gln Leu Ile Gln Ile Thr Met Lys Asp Val Asn Leu Glu Asn Val Asn Gln Gln Arg Gly Glu Lys Ser Ile Phe Lys Gly Lys Lys Ser Ser Gln Ile Ile Arg Lys Glu Asn Leu Glu Ala Met Gln Arg Pro Val Leu Leu His Leu Ile His Gly Lys Ile Lys Glu Phe Tyr Ser Tyr Gln Asn Glu Pro Ala Ala Ile Glu Asn Leu Lys Arg Gly Leu Ala Ser Leu Phe Gln Met Gln Leu Ser Ser Gly Thr Thr Asn Glu Val Asp Ile Ser Gly Asp Cys Lys Val Thr Tyr Gln Ala His Gln Asp Lys Val Thr Lys Ile Lys Ala Leu Asp Ser Cys Lys Ile Glu Arg Ala Gly Phe Thr Thr Pro His Gln Val Leu Gly Val Thr Ser Lys Ala Thr Ser Val Thr Thr Tyr Lys Ile Glu Asp Ser Phe Val Val Ala Val Leu Ser Glu Glu Ile Arg Ala Leu Arg Leu Asn Phe Leu Gln Ser Ile Ala Gly Lys Ile Val Ser Arg Gln Lys Leu Glu Leu Lys Thr Thr Glu Ala Ser Val Arg Leu Lys Pro Gly Lys Gln Val Ala Ala Ile Ile Lys Ala Val Asp Ser Lys Tyr Thr Ala Ile Pro Ile Val Gly Gln Val Phe Gln Ser Lys Cys Lys Gly Cys Pro Ser Leu Ser Glu His Trp Gln Ser Ile Arg Lys His Leu Gln Pro Asp Asn Leu Ser Lys Ala Glu Ala Val Arg Ser Phe Leu Ala Phe Ile Lys His Leu Arg Thr Ala Lys Lys Glu Glu Ile Leu Gln Ile Leu Lys Ala Glu Asn Lys Glu Val Leu Pro Gln Leu Val Asp Ala Val Thr Ser Ala Gln Thr Pro Asp Ser Leu Asp Ala Ile Leu Asp Phe Leu Asp Phe Lys Ser Thr Glu Ser Val Ile Leu Gln Glu Arg Phe Leu Tyr Ala Cys Ala Phe Ala Ser His Pro Asp Glu Glu Leu Leu Arg Ala Leu Ile Ser Lys Phe Lys Gly Ser Phe Gly Ser Asn Asp Ile Arg Glu Ser Val Met Ile Ile Ile Gly Ala Leu Val Arg Lys Leu Cys Gln Asn Gln Gly Cys Lys Leu Lys Gly Val Ile Glu Ala Lys Lys Leu Ile Leu Gly Gly Leu Glu Lys Ala Glu Lys Lys Glu Asp Ile Val Met Tyr Leu Leu Ala Leu Lys Asn Ala Arg Leu Pro Glu Gly Ile Pro Leu Leu Leu Lys Tyr Thr Glu Thr Gly Glu Gly Pro Ile Ser His Leu Ala Ala Thr Thr Leu Gln Arg Tyr Asp Val Pro Phe Ile Thr Asp Glu Val Lys Lys Thr Met Asn Arg Ile Tyr His Gln Asn Arg Lys Ile His Glu Lys Thr Val Arg Thr Thr Ala Ala Ala Ile Ile Leu Lys Asn Asn Pro Ser Tyr Met Glu Val Lys Asn Ile Leu Leu Ser Ile Gly Glu Leu Pro Lys Glu Met Asn Lys Tyr Met Leu Ser Ile Val Gln Asp Ile Leu Arg Phe Glu Thr Pro Ala Ser Lys Met Val Arg Gln Val Leu Lys Glu Met Val Ala His Asn Tyr Asp Arg Phe Ser Lys Ser Gly Ser Ser Ser Ala Tyr Thr Gly Tyr Val Glu Arg Thr Ser His Ser Ala Ser Thr Tyr Ser Leu Asp Ile Leu Tyr Ser Gly Ser Gly Ile Leu Arg Arg Ser Asn Leu Asn Ile Phe Gln Tyr Ile Glu Lys Thr Pro Leu His Gly Ile Gln Val Val Ile Glu Ala Gln Gly Leu Glu Ala Leu Ile Ala Ala Thr Pro Asp Glu Gly Glu Glu Asn Leu Asp Ser Tyr Ala Gly Leu Ser Ala Leu Leu Phe Asp Val Gln Leu Arg Pro Val Thr Phe Phe Asn Gly Tyr Ser Asp Leu Met Ser Lys Met Leu Ser Ala Ser Ser Asp Pro Met Ser Val Val Lys Gly Leu Leu Leu Leu Ile Asp His Ser Gln Glu Leu Gln Leu Gln Ser Gly Leu Lys Ala Asn Met Asp Val Gln Gly Gly Leu Ala Ile Asp Ile Thr Gly Ala Met Glu Phe Ser Leu Trp Tyr Arg Glu Ser Lys Thr Arg Val Lys Asn Arg Val Ser Val Leu Ile Thr Gly Gly Ile Thr Val Asp Ser Ser Phe Val Lys Ala Gly Leu Glu Ile Gly Ala Glu Thr Glu Ala Gly Leu Glu Phe Ile Ser Thr Val Gln Phe Ser Gln Tyr Pro Phe Leu Val Cys Leu Gln Met Asp Lys Glu Asp Val Pro Tyr Arg Gln Phe Glu Thr Lys Tyr Glu Arg Leu Ser Thr Gly Arg Gly Tyr Ile Ser Arg Lys Arg Lys Glu Ser Leu Ile Gly Gly Cys Glu Phe Pro Leu His Gln Glu Asn Ser Asp Met Cys Lys Val Val Phe Ala Pro Gln Pro Glu Ser Ser Ser Ser Gly Trp Phe (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 894 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Ile Leu Leu Ala Val Leu Phe Leu Cys Phe Ile Ser Ser Tyr Ser Ala Ser Val Lys Gly His Thr Thr Gly Leu Ser Leu Asn Asn Asp Arg Leu Tyr Lys Leu Thr Tyr Ser Thr Glu Val Leu Leu Asp Arg Gly Lys Gly Lys Leu Gln Asp Ser Val Gly Tyr Arg Ile Ser Ser Asn Val Asp Val Ala Leu Leu Trp Arg Asn Pro Asp Gly Asp Asp Asp Gln Leu Ile Gln Ile Thr Met Lys Asp Val Asn Val Glu Asn Val Asn Gln Gln Arg Gly Glu Lys Ser Ile Phe Lys Gly Lys Ser Pro Ser Lys Ile Met Gly Lys Glu Asn Leu Glu Ala Leu Gln Arg Pro Thr Leu Leu His Leu Ile His Gly Lys Val Lys Glu Phe Tyr Ser Tyr Gln Asn Glu Ala Val Ala Ile Glu Asn Ile Lys Arg Gly Leu Ala Ser Leu Phe Gin Thr Gln Leu Ser Ser Gly Thr Thr Asn Glu Val Asp Ile Ser Gly Asn Cys Lys Val Thr Tyr Gln Ala His Gln Asp Lys Val Ile Lys Ile Lys Ala Leu Asp Ser Cys Lys Ile Ala Arg Ser Gly Phe Thr Thr Pro Asn Gln Val Leu Gly Val Ser Ser Lys Ala Thr Ser Val Thr Thr Tyr Lys Ile Glu Asp Ser Phe Val Ile Ala Val Leu Ala Glu Glu Thr His Asn Phe Gly Leu Asn Phe Leu Gln Thr Ile Lys Gly Lys Ile Val Ser Lys Gln Lys Leu Glu Leu Lys Thr Thr Glu Ala Gly Pro Arg Leu Met Ser Gly Lys Gln Ala Ala Ala Ile Ile Lys Ala Val Asp Ser Lys Tyr Thr Ala Ile Pro Ile Val Gly Gln Val Phe Gln Ser His Cys Lys Gly Cys Pro Ser Leu Ser Glu Leu Trp Arg Ser Thr Arg Lys Tyr Leu Gln Pro Asp Asn Leu Ser Lys Ala Glu Ala Val Arg Asn Phe Leu Ala Phe Ile Gln His Leu Arg Thr Ala Lys Lys Glu Glu Ile Leu Gln Ile Leu Lys Met Glu Asn Lys Glu Val Leu Pro Gln Leu Val Asp Ala Val Thr Ser Ala Gln Thr Ser Asp Ser Leu Glu Ala Ile Leu Asp Phe Leu Asp Phe Lys Ser Asp Ser Ser Ile Ile Leu Gln Glu Arg Phe Leu Tyr Ala Cys Gly Phe Ala Ser His Pro Asn Glu Glu Leu Leu Arg Ala Leu Ile Ser Lys Phe Lys Gly Ser Ile Gly Ser Ser Asp Ile Arg Glu Thr Val Met Ile Ile Thr Gly Thr Leu Val Arg Lys Leu Cys Gln Asn Glu Gly Cys Lys Leu Lys Ala Val Val Glu Ala Lys Lys Leu Ile Leu Gly Gly Leu Glu Lys Ala Glu Lys Lys Glu Asp Thr Arg Met Tyr Leu Leu Ala Leu Lys Asn Ala Leu Leu Pro Glu Gly Ile Pro Ser Leu Leu Lys Tyr Ala Glu Ala Gly Glu Gly Pro Ile Ser His Leu Ala Thr Thr Ala Leu Gin Arg Tyr Asp Leu Pro Phe Ile Thr Asp Glu Val Lys Lys Thr Leu Asn Arg Ile Tyr His Gln Asn Arg Lys Val His Glu Lys Thr Val Arg Thr Ala Ala Ala Ala Ile Ile Leu Asn Asn Asn Pro Ser Tyr Met Asp Val Lys Asn Ile Leu Leu Ser Ile Gly Glu Leu Pro Gln Glu Met Asn Lys Tyr Met Leu Ala Ile Val Gln Asp Ile Leu Arg Leu Glu Met Pro Ala Ser Lys Ile Val Arg Arg Val Leu Lys Glu Met Val Ala His Asn Tyr Asp Arg Phe Ser Arg Ser Gly Ser Ser Ser Ala Tyr Thr Gly Tyr Ile Glu Arg Ser Pro Arg Ser Ala Ser Thr Tyr Ser Leu Asp Ile Leu Tyr Ser Gly Ser Gly Ile Leu Arg Arg Ser Asn Leu Asn Ile Phe Gln Tyr Ile Gly Lys Ala Gly Leu His Gly Ser Gln Val Val Ile Glu Ala Gln Gly Leu Glu Ala Leu Ile Ala Ala Thr Pro Asp Glu Gly Glu Glu Asn Leu Asp Ser Tyr Ala Gly Met Ser Ala Ile Leu Phe Asp Val Gln Leu Arg Pro Val Thr Phe Phe Asn Gly Tyr Ser Asp Leu Met Ser Lys Met Leu Ser Ala Ser Gly Asp Pro Ile Ser Val Val Lys Gly Leu Ile Leu Leu Ile Asp His Ser Gln Glu Leu Gln Leu Gln Ser Gly Leu Lys Ala Asn Ile Glu Val Gln Gly Gly Leu Ala Ile Asp Ile Ser Gly Ala Met Glu Phe Ser Leu Trp Tyr Arg Glu Ser Lys Thr Arg Val Lys Asn Arg Val Thr Val Val Ile Thr Thr Asp Ile Thr Val Asp Ser Ser Phe Val Lys Ala Gly Leu Glu Thr Ser Thr Glu Thr Glu Ala Gly Leu Glu Phe Ile Ser Thr Val Gln Phe Ser Gln Tyr Pro Phe Leu Val Cys Met Gln Met Asp Lys Asp Glu Ala Pro Phe Arg Gln Phe Glu Lys Lys Tyr Glu Arg Leu Ser Thr Gly Arg Gly Tyr Val Ser Gln Lys Arg Lys Glu Ser Val Leu Ala Gly Cys Glu Phe Pro Leu His Gln Glu Asn Ser Glu Met Cys Lys Val Val Phe Ala Pro Gln Pro Asp Ser Thr Ser Thr Gly Trp Phe (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 107 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME KEY: CDS
(B) LOCATION: 3..107 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

tt ttt ctc tgc ttc att tcc tca tat tca gct tct gtt aaa ggt cac 47 Phe Leu Cys Phe Ile Ser Ser Tyr Ser Ala Ser Val Lys Gly His aca act ggt ctc tca tta aat aat gac cga cta tac aaa ctc aca tac 95 Thr Thr Gly Leu Ser Leu Asn Asn Asp Arg Leu Tyr Lys Leu Thr Tyr tcc act gaa gtt 107 Ser Thr Glu Val (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 AMINO ACIDS
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Phe Leu Cys Phe Ile Ser Ser Tyr Ser Ala Ser Val Lys Gly His Thr Thr Gly Leu Ser Leu Asn Asn Asp Arg Leu Tyr Lys Leu Thr Tyr Ser Thr Glu Val (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

agagtccact tctca 15 (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8067 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 100..287 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 450..451 (ix) FEATURE:

(A) NAME/KEY: CDS
(B) LOCATION: 700..844 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1197..1198 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1253..1361 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1481..1482 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1586..1702 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 1764..1765 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1805..1945 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 2010..2011 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2049..2199 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 2281..2282 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2355..2512 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 2565..2566 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2595..2763 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 2871..2872 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2898..3005 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 3135..3136 (ix) FEATURE:

(A) NAME/KEY: CDS
(B) LOCATION: 3389..3601 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 3763..3764 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4077..4288 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 4386..4387 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4630..4727 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 4819..4940 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 5183..5184 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 5284..5511 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 5567..5568 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 5685..5809 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 5857..5858 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 6211..6381 (ix) FEATURE:
(A) NAME/KEY: -(B) LOCATION: 6635..6636 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 6740..8067 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 aaggttcctg agccccactg tggtagagag atgcactgat ggtgagacag catgttccct 60 tacaatgaaa actggatatg tgtcattatc tttatgcagg tcacacaact ggtctctcat 120 taaataatga ccggctgtac aagctcacgt actccactga agttcttctt gatcggggca 180 aaggaaaact gcaagacagc gtgggctacc gcatttcctc caacgtggat gtggccttac 240 tatggaggaa tcctgatggt gatgatgacc agttgatcca aataacggtg ggcattttct 300 accagataaa tgcaaagatt agatatcaga agtttttgga gaagtgtacc attggacagc 360 acttgtattg ggttcccgtt tataatccat tagtttctta tcttatcact aaaacaagca 420 ggtctttgtt ttaaggtttg gtgatgaaag ttattttaag cctaaagtca cagagttctt 480 taagtattgc tatttttgcc ttattaaaaa acctagttta taaatacctt ctccattctt 540 ttaaagtgag tggcaaggtc ctataaatca tgaattgaaa aatgacagaa gaaattgtgg 600 ccaactcttt ctgtttcttt atcattttat tttcagagat actctgatga agacagatat 660 aggaagtttt ttttaacagc tttctttctg ttactccaga tgaaggatgt aaatgttgaa 720 aatgtgaatc agcagagagg agagaagagc atcttcaaag gaaaaagccc atctaaaata 780 atgggaaagg aaaacttgga agctctgcaa agacctacgc tccttcatct aatccatgga 840 aaggtaaagg ggcctttaga ttccacaact ttttctccaa cttcatattt ttcttccctt 900 cagtagatat tattttgagg taatcacatt gtaactactt ttatggtaaa tggaatttct 960 tcaagaacta aagaacagag gttgtaaatt aaatgtttcc aaactgaatc aatgccctga 1020 gttcccttac atttactagc caatttgttt cctatttttc tggaaatctt tatagtggaa 1080 tgaagtattt atttattgat gaaaggcatt attaaaaggt aaatttctca tcaaattata 1140 agggattaca aacataatgt aacaaagcaa gtcatcaaag catgattgga tgaattctct 1200 gataaatgat gcatttttgc ttcatttgtg ttctgttccc ctctccccac caggtcaaag 1260 agttctactc atatcaaaat gaggcagtgg ccatagaaaa tatcaagaga ggtctggcta 1320 gcctatttca gacacagtta agctctggaa ccaccaatga ggtacttacc aatattaata 1380 aggattcagc atctcaataa aatttgtaag gatttctact tatacaattt cagtagaaga 1440 gttactacta aggtaatgct cagaaaaggt gacttgtgta gtcccctatg gcctattaga 1500 gacctcaatt ttcaagccac ttctcactag aattcaaatg gcccacaagg aatcccaagc 1560 attatgccct tgcctttctt tttaggtaga tatctctgga aattgtaaag tgacctacca 1620 ggctcatcaa gacaaagtga tcaaaattaa ggccttggat tcatgcaaaa tagcgaggtc 1680 tggatttacg accccaaatc aggtatgata gatgtcactt tctttgaggc attaaaataa 1740 ttacattttg tagagactaa tttacgatga ttacttgtta taaagatggc tatttattta 1800 tttaggtctt gggtgtcagt tcaaaagcta catctgtcac cacctataag atagaagaca 1860 gctttgttat agctgtgctt gctgaagaaa cacacaattt tggactgaat ttcctacaaa 1920 ccattaaggg gaaaatagta tcgaagtaag ataatgctaa aatttttatt ttctttgcta 1980 ttctttgtta tattattata cttgatttgt atgattataa tatagcattt ccctttggta 2040 ttatgcaggc agaaattaga gctgaagaca accgaagcag gcccaagatt gatgtctgga 2100 aagcaggctg cagccataat caaagcagtt gattcaaagt acacggccat tcccattgtg 2160 gggcaggtct tccagagcca ctgtaaagga tgtccttctg taagtgcaga caaatatggg 2220 aataatcatg acatcagact ctgttttcat tttgtctcca gtgaaagcat caactcattc 2280 aggagaacac cctttgtaaa tgtggatgtt cacagttatg agtggggtat gagcctgcag 2340 tgtatgtttt gcagctctcg gagctctggc ggtccaccag gaaatacctg cagcctgaca 2400 acctttccaa ggctgaggct gtcagaaact tcctggcctt cattcagcac ctcaggactg 2460 cgaagaaaga agagatcctt caaatactaa agatggaaaa taaggaagta ttgtaagttc 2520 cccaaccttt gtgtggggtt gtctgtcaga aacatttctg gagtggatat ccatgattat 2580 gccttttttt atagacctca gctggtggat gctgtcacct ctgctcagac ctcagactca 2640 ttagaagcca ttttggactt tttggatttc aaaagtgaca gcagcattat cctccaggag 2700 aggtttctct atgcctgtgg atttgcttct catcccaatg aagaactcct gagagccctc 2760 attgtaagtc aaatagaaaa taaagaccct caactcctat aaaacttctt aagaatatta 2820 acagtaatta aaagtttctt agatccgaat tcttcgccct atagtgagtc actattttat 2880 ccctgggtgg ttaatagagt aagttcaaag gttctattgg tagcagtgac atcagagaaa 2940 ctgttatgat catcactggg acacttgtca gaaagttgtg tcagaatgaa ggctgcaaac 3000 tcaaagtaag tgcaaatcca atctcatgta ttacatcatt ctacaccatt gtccatttga 3060 tactcaccat gctgcctact attggcactc ctaattctct ttactctatt ctacttacct 3120 tatttgnata gcaataacac aatatgccca ttattgataa tactcattgc ttcttaagaa 3180 tgtatatgta ttttttttaa aaaaagcata acacctttat caagctttac ttgtttgctt 3240 ttattccact gtgtgcctca gtcaagcaac caatgcaaaa ctttgtaaaa ctgtaggttg 3300 ctttcttgga cccaagaata aagccagtct cacccaagtc ttcttcaatg tatggtcatg 3360 catatatcta aggtatatga tttttcaggc agtagtggaa gctaagaagt taatcctggg 3420 aggacttgaa aaagcagaga aaaaagagga caccaggatg tatctgctgg ctttgaagaa 3480 tgccctgctt ccagaaggca tcccaagtct tctgaagtat gcagaagcag gagaagggcc 3540 catcagccac ctggctacca ctgctctcca gagatatgat ctccctttca taactgatga 3600 ggtaaaatct ccaagaatat ttgcaacatt tacagaagaa aaaaaaaaag catgctgaac 3660 atgagtcaaa tgcaaattcc gctcaagtca ctctgtattt tccccaaata gtcttctctc 3720 ctgcttaaaa ataactctta aattgcattt ggggctattc taaatgttta atttctcagg 3780 ctatgcctaa tgtgcataag gaagtatgtg gtctgaagtt cactacagtc atggaagaaa 3840 gagatggaga aagccaccag ctcttaacgg cctcagccta gaagtgatcc tcatagattc 3900 tatccatggc gtattagcca gaactagtca cgtggccccc accaaatcac aaaggaatct 3960 gggaaatgta gtaacacatg tatattttta tgaacactca ctattcctgc tattcctgct 4020 gaaatgtcca ttttaaaaat ctagatgtgc actaagtttg aacatcttat gaacaggtga 4080 agaagacctt aaacagaata taccaccaaa accgtaaagt tcatgaaaag actgtgcgca 4140 ctgctgcagc tgctatcatt ttaaataaca atccatccta catggacgtc aagaacatcc 4200 tgctgtctat tggggagctt ccccaagaaa tgaataaata catgctcgcc attgttcaag 4260 acatcctacg ttttgaaatg cctgcaaggt ataatacatt gcacatgtct ctctgtgtat 4320 tcaagcttat ttgtgtgttc atggggtacc gatgtagcta ataataatga tgtggtcatt 4380 atgcaaagct ggacaccctt gccttgctgt cattttgata gcaaactaaa tttcaaatat 4440 ctgagtaatg aaggggctag ccctaatcct gatgctacca cgccagctgg caccaccctg 4500 gctcttggaa aggcatgagg aaaatttggc ttcctctttt ttccactgag gatttttttt 4560 ttccaaattt gacttgggaa acagtcatta caatgaatgt gcagcttttt ttttcctcat 4620 atgttgcagc aaaattgtcc gtcgagttct gaaggaaatg gtcgctcaca attatgaccg 4680 tttctccagg agtggatctt cttctgccta cactggctac atagaacgta tgtacaccaa 4740 aaagaggttc tccttccata ccccacaact tagcattgct ggaactgcta ttaaattaca 4800 gttattgtgt gtcatcaggt agtccccgtt cggcatctac ttacagccta gacattctct 4860 actcgggttc tggcattcta aggagaagta acctgaacat ctttcagtac attgggaagg 4920 ctggtcttca cggtagccag gtaactcact tctcatggat tttgcttaat aaagtatgca 4980 agaaatcagg ctgaggtaaa ataaaacata tatgctgtgg gtaatgctat agaatgtata 5040 agttaatggt ggcttctgtc atattttgcc catgatttcc ttatctgtaa gaggctgtat 5100 ggtttatagt cactcagaga aagtttcgaa tttgaacttg aaacctaagt aatttgatcc 5160 attgaacttg acaaatgtcc atttggcccc ttgagaagtt ctagctgcag ctcagaagct 5220 tcaccattat ttacagagca ggcagggagc ttgcgtcatg aacattatat tgattttatc 5280 caggtggtta ttgaagccca aggactggaa gccttaatcg cagccacccc tgacgagggg 5340 gaggagaacc ttgactccta tgctggtatg tcagccatcc tctttgatgt tcagctcaga 5400 cctgtcacct ttttcaacgg atacagtgat ttgatgtcca aaatgctgtc agcatctggc 5460 gaccctatca gtgtggtgaa aggacttatt ctgctaatag atcattctca ggtaattcan 5520 ycagtctgtg agtatttatt gagtccctaa actacgccag gcacgtaatc aacacaactc 5580 aaatggaatt atctacagca ggaggtcaaa tgtnccattg gaaagggggt taactaaatt 5640 gtacttatta tttttataac tattattatg cttttttctt ctaggaactt cagttacaat 5700 ctggactaaa agccaatata gaggtccagg gtggtctagc tattgatatt tcaggtgcaa 5760 tggagtttag cttgtggtat cgtgagtcta aaacccgagt gaaaaatagg taagtgttta 5820 tgcattatac atttatgaat tacatataag actatatctt gggtatttct gacctgctga 5880 gaggacctgg gttccaagaa tgtttttcat tttggtcttt gttatgccca tacgaaacaa 5940 tgtagtatct tacagacact ccccacatct gcaactgaag gcaggggaga gctcagggga 6000 agggcaaacc ttccctgccc aatatctgag actcaccagg ccctggttac cagcagaact 6060 ctaagcacat ccaggtcacc tctgaatccc ttaagtgttt ccttccagtc actggcatca 6120 tacgttcaga ccctgtaaag ttacagctgt tagtccaata ccattaaata taatatgaac 6180 aagttttttc tttttttctc aaatgtttag ggtgactgtg gtaataacca ctgacatcac 6240 agtggactcc tcttttgtga aagctggcct ggaaaccagt acagaaacag aagcaggttt 6300 ggagtttatc tccacagtgc agttttctca gtacccattc ttagtttgca tgcagatgga 6360 caaggatgaa gctccattca ggtaagatgc agcgtacagg tcatgttcca ggaccatccc 6420 cagtgcacca ggaacttgca ttcagtttag aacattcagt ttcagaatta aaacaaaaca 6480 gtagaaaccc agggaaagat gaattttctt taaatgagta gaagaataat tgataaggcc 6540 aaaaaaagtc agtttctggg ataccaaaaa aaaatctaat gactagttca tgtgattctg 6600 gagatagtta tcatattcta atccagaaac aattttgctt tggaacagaa acttcaagta 6660 cattcagtaa cttggctgga gaggtatagg gtgacttaac tgtgtgtgta attctgttaa 6720 tgttgctgtt gttgtacagg caatttgaga aaaagtacga aaggctgtcc acaggcagag 6780 gttatgtctc tcagaaaaga aaagaaagcg tattagcagg atgtgaattc ccgctccatc 6840 aagagaactc agagatgtgc aaagtggtgt ttgcccctca gccggatagt acttccagcg 6900 gatggttttg aaactgacct gtgatatttt acttgaattt gtctccccga aagggacaca 6960 atgtggcatg actaagtact tgctctctga gagcacagcg tttacatatt tacctgtatt 7020 taagattttt gtaaaaagct acaaaaaact gcagtttgat caaatttggg tatatgcagt 7080 atgctaccca cagcgtcatt ttgaatcatc atgtgacgct ttcaacaacg ttcttagttt 7140 acttatacct ctctcaaatc tcatttggta cagtcagaat agttattctc taagaggaaa 7200 ctagtgtttg ttaaaaacaa aaataaaaac aaaaccacac aaggagaacc caattttgtt 7260 tcaacaattt ttgatcaatg tatatgaagc tcttgatagg acttccttaa gcatgacggg 7320 aaaaccaaac acgttcccta atcaggaaaa aaaaaaaaaa aaaaggtagg acacaaccaa 7380 cccatttttt ttctcttttt ttggagttgg gggcccaggg agaagggaca agacttttaa 7440 aagacttgtt agccaacttc aagaattaat atttatgtct ctgttattgt tagttttaag 7500 ccttaaggta gaaggcacat agaaataaca tctcatcttt ctgctgacca ttttagtgag 7560 gttgttccaa agacattcag gtctctacct ccagccctgc aaaaatattg gacctagcac 7620 agaggaatca ggaaaattaa tttcagaaac tccatttgat ttttcttttg ctgtgtcttt 7680 ttgagactgt aatatggtac actgtcctct aagggacatc ctcattttat ctcacctttt 7740 tgggggtgag agctctagtt catttaactg tactctgcac aatagctagg atgactaaga 7800 gaacattgct tcaagaaact ggtggatttg gatttccaaa atatgaaata aggaaaaaaa 7860 tgtttttatt tgtatgaatt aaaagatcca tgttgaacat ttgcaaatat ttattaataa 7920 acagatgtgg tgataaaccc aaaacaaatg acaggtcctt attttccact aaacacagac 7980 acatgaaatg aaagtttagc tagcccacta tttgtaaatt gaaaacgaag tgtgataaaa 8040 taaatatgta gaaatcatat tgaattc 8067 (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ggcactggat gcagttgagg attgct 26 (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ggtcaatatg attcttcttg ctgtgc 26 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

ccggaattcc ctaccaggct catcaagaca aag 33 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

acggccattc ccattgtggg gcaggt 26 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

tgacacccaa gacctgattt ggggtc 26 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

gcctgcttcg gttgtcttca gctct 25 (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

cgcggatcct tctgacagcc tcagccttgg a 31 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

gggagatcat atctctggag agcagt 26 (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

cggcggatcc agcataggag tcaaggttct c 31 (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

cccttacaat gaaaactgg 19 (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

ggtacacttc tccaaaaact t 21 (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

agg aat cct gat ggt gat gat gac cag ttg atc 33 Arg Asn Pro Asp Gly Asp Asp Asp Gln Leu Ile (2) INFORMATION FOR SEQ ID NO : 21 :

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Arg Asn Pro Asp Gly Asp Asp Asp Gln Leu Ile (2) INFORMATION FOR SEQ ID NO:22:
.(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..27 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

agg aat ctg atg gtg atg atg acc agt tgatg 32 Arg Asn Leu Met Val Met Met Thr Ser (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Arg Asn Leu Met Val Met Met Thr Ser (2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 302 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:

(A) NAME/KEY: exon (B) LOCATION: 106..203 (ix) FEATURE:
(A) NAME/KEY: mutation (b) LOCATION: replace (119,"") (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

atttggcttc ctcttttttc cactgaggat ttttttttcc aaatttgact tgggaaacag 60 tcattacaat gaatgtgcag cttttttttt cctcatatgt tgcagcaaaa ttgtccgtcg 120 agttctgaag gaaatggtcg ctcacaatta tgaccgtttc tccaggagtg gatcttcttc 180 tgcctacact ggctacatag aaggtatgta caccaaaaag aggttctcct tccatacccc 240 acaacttagc attgctggaa ctgctattaa attacagtta tagtgtgtca tcaggtagtc 300 cc (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

ctctaccagc gagtattaat 20 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

acgtaggatg tcttggacaa tggagagcat gta 33 (2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

gatcagttgg ttatcatcac catcaggact 30 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Met Ile Leu Leu Ala Val Leu Phe Leu Cys Phe Ile (2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

ggtcaatatg attcttcttg ctgtgc 26 (2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

gcctcgatac tattttgcct gct 23

Claims

What is claimed is:

1. A nucleic acid molecule comprising a nucleic acid sequence (i) coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein and (ii) having the nucleotide sequence shown in SEQ ID NOS: 1, 2, or 8, 1 together with 5, 2 together with 7, the first 108 bases of 2 together with 8, or the first 108 bases of 2 together with 7 and 8.

2. The nucleic acid molecule according to claim 1 which is a DNA
molecule and wherein the nucleic acid sequence is a DNA sequence.

3. A DNA molecule comprising a DNA sequence which (a) is complementary to the DNA sequence according to claim 2; or (b) hybridizes to the complement of the DNA sequence according to claim 2 and encodes a protein having the biological activity of the high molecular weight subunit of microsomal triglyceride transfer protein, wherein hybridization comprises a final wash at 65°C in 1 × SSC, 0.1% SDS; or (c) is degenerated to either of the sequences according to claim 2, or 3(b).

4. An expression vector comprising the DNA sequence according to any one of claims 1 to 3 wherein said DNA sequence is under the control of regulatory elements suitable to direct expression in an appropriate host cell.

5. A prokaryotic or eukaryotic host cell comprising the expression vector according to claim 4.

6. A method for producing a polypeptide molecule having all or part of the high molecular weight subunit of microsomal triglyceride transfer protein, which comprises culturing a host cell according to claim 5 under conditions permitting expression of the polypeptide.

7. A method for detecting a nucleic acid sequence according to any one of claims 1 to 3 coding for all or part of the high molecular weight subunit of microsomal triglyceride transfer protein or a related nucleic acid sequence, which comprises:
(a) contacting the nucleic acid sequence with a detectable marker which binds specifically to at least part of the nucleic acid sequence, and (b) detecting the marker so bound;
wherein the presence of bound marker indicates the presence of the nucleic acid sequence.

8. The method according to claim 7 wherein the detectable marker is a nucleotide sequence of at least about 15 nucleotides in length complementary to at least a portion of the nucleic acid sequence coding for the high molecular weight subunit of microsomal triglyceride transfer protein.

9. The method according to claim 8 wherein the nucleotide sequence is a genomic DNA sequence, a cDNA sequence, an RNA sequence, a sense RNA
sequence or an antisense RNA sequence.

10. The method according to claim 9, wherein the detectable marker is labelled with a radioisotope and the detecting step is carried out by autoradiography.

11. The nucleic acid molecule according to any one of claims 1 to 3 or the high molecular weight subunit of microsomal triglyceride transfer protein encoded by said nucleic acid molecule for use in therapy or diagnosis of a disease or disorder associated with an increased amount or activity of microsomal triglyceride transfer protein.

12. A pharmaceutical composition for stabilizing or causing regression of atherosclerosis, decreasing serum lipid levels, preventing or treating pancreatitis, or preventing or treating obesity, in a mammal, said composition comprising the nucleic acid molecule according to any one of claims 1 to 3 or the high molecular weight subunit of microsomal triglyceride transfer protein encoded by said nucleic acid molecule, and a pharmaceutically acceptable carrier.

13. Use of a therapeutically effective amount of (a) an inhibitor of microsomal triglyceride transfer protein activity or (b) the DNA molecule of claim 3(a) which decreases the amount of microsomal triglyceride transfer protein for the preparation of a pharmaceutical composition for preventing, stabilizing or causing regression of atherosclerosis in a mammalian species, wherein said inhibitor is 1-[3-(6-fluoro-1-tetralanyl)methyl]-4-O-methoxyphenyl piperazine).

14. Use of a therapeutically effective amount of (a) an inhibitor of microsomal triglyceride transfer protein activity or (b) the DNA molecule of claim 3(a) which decreases the amount of microsomal triglyceride transfer protein for the preparation of a pharmaceutical composition for decreasing serum lipid levels in a mammalian species, wherein said inhibitor is 1-[3-(6-fluoro-1-tetralanyl)methyl]-4-O-methoxyphenyl piperazine).

15. The use of claim 14, wherein the lipid is cholesterol, triglyceride, cholesteryl ester, or phosphatidylcholine.

16. Use of a therapeutically effective amount of (a) an inhibitor of microsomal triglyceride transfer protein activity or (b) the DNA molecule of claim 3(a) which decreases the amount of microsomal triglyceride transfer protein for the preparation of a pharmaceutical composition for preventing or treating pancreatitis in a mammalian species, wherein said inhibitor is 1-[3-(6-fluoro-1-tetralanyl)methyl]-4-0-methoxyphenyl piperazine).

17. Use of a therapeutically effective amount of (a) an inhibitor of microsomal triglyceride transfer protein activity or (b) the DNA molecule of claim 3(a) which decreases the amount of microsomal triglyceride transfer protein for the preparation of a pharmaceutical composition for preventing or treating obesity in a mammalian species, wherein said inhibitor is 1 -[3-(6-fluoro-1-tetralanyl)methyl]-4-O-methoxyphenyl piperazine).

18. A process for the preparation of a nucleic acid molecule or a DNA
molecule according to any one of claims 1 to 3 or 11 which comprises using any of the following techniques known in the art:
(a) screening a genomic or cDNA library for the respective DNA
sequence and isolating it, or (b) chemically synthesizing the DNA sequence, or (c) synthesizing the DNA sequence by the polymerase chain reaction (PCR).

19. A process for the preparation of the pharmaceutical composition of claim 12 which comprises combining the nucleic acid molecule or the protein with the pharmaceutically acceptable carrier.