Compositions and methods for detecting pancreatic disease

Methods for screening a patient for pancreatic disease are disclosed and are based upon detection of a mutation in the gene encoding insulin promoter factor-1 (IPF-1) which is linked to diabetes mellitus and pancreatic agenesis.

FIELD OF THE INVENTION
 The invention relates to the fields of clinical diagnosis and of screening
 for risk of genetically-determined disorders relating to pancreatic
 disease.
 BACKGROUND OF THE INVENTION
 Pancreatic disorders, particularly diabetes mellitus, constitute a major
 public health problem, as they affect a sizeable proportion of the
 population and have a profound negative impact on the overall health and
 quality of life of those individuals so afflicted. While a variety of
 pharmaceutical compositions have been developed to compensate for
 reductions in insulin production brought about by pancreatic deficiency,
 modifications in lifestyle, such as exercise, adherence to a sensible
 diet, avoidance of tobacco and moderation of alcohol consumption, can do
 much to alleviate its effects; therefore, it is desirable to conduct
 genetic screening to identify individuals who are predisposed toward the
 development of diabetes and other pancreatic disorders while they are
 still asymptomatic.
 Maturity onset (type II) diabetes mellitus is a highly prevalent disease
 caused by an imbalance between insulin production by the endocrine
 pancreatic .beta.-cells and the insulin requirements of peripheral
 tissues. This results in hyperglycemia and secondary cardiovascular,
 renal, ocular, and neurological complications. Susceptibility to type II
 diabetes is generally believed to be inherited as a complex polygenic
 trait. However, a distinct subset of early onset type II diabetes
 (maturity onset diabetes of the young: MODY) is transmitted as an
 autosomal dominant monogenic disorder (Tattersall and Fajans, 1975,
 Diabetes, 24: 44-53). The identification of genes implicated in MODY can
 be regarded as an effective strategy to gain insight into the molecular
 pathogenesis of the more common and complex late onset forms of type II
 diabetes mellitus. To date, three distinct MODY genetic loci have been
 identified (Froguel et al., 1992, Nature, 356: 162-164), two of which
 correspond to transcription factors expressed in pancreatic .beta.-cells
 [HNF1.alpha. (MODY3) and HNF4.alpha. (MODY1)]. MODY2 is caused by
 mutations in the glucokinase gene.
 Insulin promoter factor-1 (IPF-1) is a transcription factor known to
 mediate glucose-responsive stimulation of insulin gene expression and is
 necessary for pancreas development. This homeodomain protein, also known
 as IDX-1, STF-1 and PDX-1, is critical for development of the pancreas in
 mice and is a key factor for the regulation of the insulin gene in the
 .beta.-cells of the endocrine pancreas (Miller et al., 1994, EMBO J., 13:
 1145-1156; Leonard et al., 1993, Mol. Endocr., 7: 1275-1283; Ohlsson et
 al., 1993, EMBO J. 12: 4251-4259; Jonnson et al., 1994, Nature, 371:
 606-609; Peshavaria et al., 1994, Mol. Endocr. 8: 806-816; Peers et al.,
 1994, Mol. Endocr.. 8: 1798-1806). Targeted disruption of the Ipf1 gene
 encoding IPF-1 in transgenic mice results in a failure of the pancreas to
 develop (pancreatic agenesis), although heterozygosity for this mutation
 and the wild-type allele has no apparent deleterious consequence (Jonnson,
 1994, supra; Offield et al., 1996, Development, 122: 983-995). An object
 of the present invention comprises a screening assay by which to assess a
 patient's risk of developing MODY4, to distinguish between MODY4 and other
 forms of MODY and to assist in determining the genetic basis for other
 pancreatic disorders that might result from IPF-1 deficiency.
 SUMMARY OF THE INVENTION
 The invention is based on the discovery that a mutation of the IPF1 gene
 (herein referred to as an "IPF1 mutation"), which encodes the insulin
 promoter factor-1 (IPF-1) transcription factor, is indicative of
 pancreatic disease, in particular of form early onset type II diabetes and
 of pancreatic agenesis.
 By "IPF1 mutation", we mean any defect in the IPF1 gene or regulatory
 regions (promoter, intron, splice sites) which leads to a loss of
 biological activity of the native IPF-1 protein or to a novel or altered
 IPF-1 protein function. Such activity includes, but is not limited to, the
 transcriptional activation of a gene whose regulatory sequences comprise
 an IPF-1 binding motif, as defined herein.
 As used herein, "pancreatic disease" is defined as encompassing the
 absence, underdevelopment or maldevelopment of the pancreas, or loss or
 impairment of pancreatic function in terms of either the production,
 storage, stability or secretion of factors such as proteins, lipids,
 carbohydrates or other messenger molecules essential for the homeostatic
 regulation of pancreatic functions. Such factors might include hormones,
 enzymes, fatty acids, proteins or derivatized proteins such as
 lipoproteins, glycoproteins or phosphoproteins. Specifically, "pancreatic
 disease" is used to denote pancreatic agenesis or malformation, diabetes
 or any other manifestation of disease due to malfunctioning of the
 pancreas.
 "Mature onset diabetes of the young" or "MODY" is a term which refers to a
 particular subset of cases of type II diabetes, namely those in which the
 patient becomes diabetic at an early age, typically before age 25, but in
 which fasting hyperglycemia, if present, can be regulated without insulin
 for at least two years following onset of clinical symptoms and in which
 genetic transmission of the disease is by an autosomal dominant mechanism.
 "MODY4" refers to MODY that has an IPF1 mutation as its underlying genetic
 cause.
 "IPF-1 binding motif" is defined as any gene regulatory sequence to which
 IPF-1 binds specifically in order to regulate transcription of the
 associated gene. Such sequences include, but are not limited to: FAR-FLAT
 (also called INS1-FLAT), the Far-linked AT-rich element of the rat insulin
 1 gene (5'- GATCCTTCTTAATCTAATTACCCTAGGTCTAA-3') [SEQ ID NO: 17];
 SMS-TAAT1, a FLAT-like element 438 to 461 nucleotides upstream of the rat
 somatostatin gene (5'-GATCCCTGATTGCATATTAATTCTCAGATA-3') [SEQ ID NO: 18];
 SMS-TAAT2, a FLAT-like element 290 to 303 nucleotides upstream of the rat
 somatostatin gene (5'-GATCCGATCTCAGTAATTAATCATGCACCA-3') [SEQ ID NO: 19];
 SMS-UE-B, the B domain of the rat somatostatin upstream enhancer
 (5'-GATCCGCGAGGCTAATGGTGCGTAAAAGCACTGGTGA-3') [SEQ ID NO: 20]; and SMS-PS,
 a transcriptional silencer element 219-233 nucleotides upstream of the rat
 somatostatin gene (5'-GATCCAGGCAAGATTATTTGGTCA-3') [SEQ ID NO: 21].
 The invention comprises a procedure for screening for pancreatic disease in
 a patient, comprising performing a detection step for a mutation in the
 gene encoding insulin promoter factor 1, wherein detection of a mutation
 is indicative of pancreatic disease.
 In a preferred embodiment, the further step is carried out of obtaining a
 positive result in which said patient is homozygous for a mutation in
 IPF1, homozygosity being indicative of pancreatic agenesis.
 It is preferable that a further step is carried out of obtaining a positive
 result in which said patient is heterozygous for a mutation in IPF1,
 heterozygosity being indicative of the presence of diabetes mellitus, more
 preferable that said diabetes mellitus is of the form early onset type II
 and most preferable that said diabetes mellitus of said form early onset
 type II is mature onset diabetes of the young (MODY).
 Preferably, the mutation in IPF1 is a deletion of a single base pair, more
 preferably, the mutation results in a translational frame shift, and most
 preferably, the mutation is IPF1.DELTA.C (also denoted Pro63fs.DELTA.C or
 Pro63fsdelC).
 A further object of the present invention encompasses a method for
 screening a patient for a mutation in the gene encoding insulin promoter
 factor 1 (IPF-1), comprising the steps of providing a nucleic acid sample
 from said patient and detecting a mutation in said gene in said sample,
 wherein detection of a mutation is indicative of pancreatic disease.
 It is contemplated that the family of said patient comprises individuals
 affected with pancreatic disease, that said patient is asymptomatic with
 regard to pancreatic disease.
 In another preferred embodiment, said patient is afflicted with symptoms of
 pancreatic disease. Preferably, said symptoms of pancreatic disease are
 those of diabetes mellitus, more preferably of said diabetes mellitus of
 the form early onset type II, and most preferably of mature onset diabetes
 of the young (MODY).
 It is contemplated that said nucleic acid sample used according to the
 inventive method is genomic DNA, and preferred that the method comprise a
 step, after the step of providing said sample, wherein a PCR product of
 the IPF1 gene of said genomic DNA is generated for use in said detection
 step.
 It is also contemplated that said nucleic acid sample used according to the
 inventive method is mRNA, and preferred that the method comprise the
 steps, after the step of providing said sample, wherein a reverse
 transcript of said mRNA is generated and a PCR product of the IPF1 gene is
 made from said reverse transcript for use in the said detection step.
 It is preferred that said detection step using said PCR product comprises
 DNA sequencing or single-strand conformation polymorphism analysis.
 A further object of the present invention is a method for screening a
 plurality of patients for a mutation in the gene encoding insulin promoter
 factor 1 (IPF-1) comprising the steps of providing a plurality of nucleic
 acid samples from a corresponding plurality of patients and detecting a
 mutation in said plurality of samples, wherein detection of said mutation
 in said sample is indicative of pancreatic disease in a said corresponding
 patient.
 It is preferred that said nucleic acid sample is genomic DNA.
 It is contemplated that said plurality of patients comprises a group of
 unrelated individuals. It is preferred that said plurality of patients
 comprises a group of related individuals.
 It is additionally preferred that said mutation in the gene encoding IPF-1
 is detected in a patient selected from said plurality of patients who is
 asymptomatic with regard to pancreatic disease.
 It is preferable that said pancreatic disease is diabetes mellitus, more
 preferable that said diabetes mellitus is of the form early onset type II,
 highly preferable that said diabetes of the form early onset type II is
 MODY, and most preferable that said MODY is of the type MODY4.
 It is also contemplated that said mutation is detected in said gene
 encoding IPF-1 in a patient selected from said plurality of patients who
 shows observable clinical manifestations of pancreatic disease.
 It is preferable that said pancreatic disease is diabetes mellitus, more
 preferable that said diabetes mellitus is of the form early onset type II
 and highly preferable that said diabetes of the form early onset type II
 is MODY.
 Most preferably, said patient selected form said plurality of patients who
 shows observable clinical manifestations of MODY has MODY4.
 Another object of the present invention is a method of testing a patient
 who is afflicted with MODY to determine whether or not said patient has
 MODY4, comprising performing a detection step for a mutation in the gene
 encoding insulin promoter factor 1, wherein detection of a mutation is
 indicative of MODY4.
 The invention also encompasses a method of screening for pancreatic disease
 in a patient whose parents both carry an inactivating mutation in the gene
 encoding IPF-1, comprising performing a detection step for a mutation in
 the gene encoding IPF-1, wherein detection of a said mutation is
 indicative of pancreatic disease.
 It is preferred that the method further comprises the step of obtaining a
 result in which said patient is heterozygous for said mutation,
 heterozygosity being indicative of MODY4.
 It is also preferred that the method further comprises the step of
 obtaining a result in which said patient is homozygous for said mutation,
 homozygosity being indicative of pancreatic agenesis.
 Preferably, said testing according to said method is prenatal.
 It is also contemplated that said testing according to said method is
 postnatal.
 The invention also encompasses a method for testing in a patient both
 alleles of the gene encoding IPF-1 for a mutation, comprising the steps of
 providing a nucleic acid sample from said patient performing a detection
 step for a mutation in said gene encoding IPF-1 and detecting a mutation
 in one or both allelic copies of said gene in said sample wherein
 homozygosity of a mutation is indicative of pancreatic agenesis and
 heterozygosity of a mutation is indicative of MODY4.
 Preferably, the detection steps of said method comprise the steps of
 hybridizing to said nucleic acid sample equimolar amounts of labeled
 oligonucleotide probes unique to wild-type and mutant IPF1 sequences under
 conditions that permit specific hybridization of each to its target
 sequence, and comparing quantitatively the extent of hybridization of the
 two probes to molecules present in said nucleic acid sample, wherein
 non-hybridization of the wild-type probe indicates homozygosity for the
 mutant allele and non-hybridization of the mutant probe indicates
 homozygosity for the wild-type, while a 1:1 ratio of hybridization of
 wild-type and mutant probes indicates heterozygosity for the two alleles
 at said locus.
 In a preferred embodiment, said testing is performed prenatally.
 It is also preferred that said testing is performed postnatally.
 In another preferred embodiment, the detection steps of said method
 comprise the steps of amplifying by PCR a region of said gene encoding
 IPF-1 to make amplified products that encompass said mutation, cloning the
 products of said PCR and performing DNA sequence analysis on multiple,
 independent clones resulting from said cloning to detect the presence or
 absence of a mutation in each such clone, wherein the failure to detect
 more than one sequence from among the clones resulting from a given sample
 is indicative of homozygosity at that locus.
 It is also preferred that the detection steps of said method comprise the
 steps of amplifying by PCR a region of said gene encoding IPF-1 to make
 amplified products that encompass said mutation and performing
 single-strand comformation polymorphism (SSCP) analysis on said amplified
 products, wherein observation of one conformer is indicative of
 homozygosity and two conformers is indicative of heterozygosity at said
 locus.
 Another object of the present invention is an isolated nucleic acid
 comprising the nucleotide sequence of SEQ ID NO: 1 with a cytosine
 deletion at any one of bases 202 through 207.

DETAILED DESCRIPTION OF THE INVENTION
 We have developed a molecular genetic screening assay for pancreatic
 disease in a patient or for risk of pancreatic disease based upon
 detection of inactivating mutations in the gene encoding the insulin
 promoter factor-1, a transcription factor that mediates pancreatic
 development and function. This assay comprises detection of a mutation in
 a nucleic acid sample derived from a peripheral blood sample drawn from a
 patient, as described below.
 Pancreatic agenesis in humans is a rare disorder; only 8 cases have been
 reported in the literature (Wright et al., 1993, Am. J. Dis. Child., 147:
 607-609; Howard et al., 1980, J. Pediat. 97: 786-789; Widnes et al., 1982,
 J. Pediat. 100: 926; Lemons et al., 1979, Pediatrics, 64: 255-257; Mehes
 and Vamosk, 1976, Acta Paediat. Acad. Sci. Hung., 17: 175-176; Dourov and
 Buyl-Strouvens, 1969, Arch. Fr. Pediat., 26: 641-650; Sherwood et al.,
 1995, Pediat. Res., 8: 360). Recently, a female Caucasian infant was
 described in which the diagnosis of pancreatic agenesis was made shortly
 after birth and appropriate therapy was instituted (Wright et al., 1993,
 supra). The infant was underweight for gestational age and presented with
 neonatal diabetes mellitus at birth and, at age 18 days, with pancreatic
 exocrine insufficiency. By ultrasound examination, the pancreas was
 absent. After replacement of insulin and pancreatic enzymes was begun, she
 developed normally and continues to do well at 5 years of age. As
 described in Example 1, we performed a genetic screening assay on this
 patient for a mutation in the gene encoding IPF-1 and found that the
 congenital pancreatic agenesis in this subject was attributable to
 homozygosity for an inactivating mutation in exon 1 of the protein coding
 sequence of the IPF1 gene (IPF1.DELTA.C) (Stoffers et al., 1997, Nature
 Genetics, 15: 106-110). Example 2 describes allele-specific hybridization
 analysis of IPF1, while Example 3 presents single-strand conformation
 polymorphism analysis, both for the purpose of either detecting a mutation
 or determining its dosage in the patient. In Example 4, use of the present
 invention in the detection of a mutation in IPF1 in a plurality of
 individuals, whether in the course of population studies or in pedigree
 analysis, is demonstrated. Example 5 illustrates use of the invention to
 assign linkage of a case of MODY to a particular genetic locus. In Example
 6, an extended haplotype analysis using IPF1 and closely-linked
 chromosomal markers is presented. In Example 7, a C-terminal translation
 product of the mutant IPF1 gene is examined.
 METHODS OF THE INVENTION
 Methods enabling use of the present invention are described in detail in
 Examples 1 and 2. They include the polymerase chain reaction, DNA sequence
 analysis, allele-specific hybridization, single-strand conformation
 polymorphism analysis, mammalian cell culture and transfection, in vitro
 protein expression, Western blotting, Northern analysis, reverse
 transcription, Southern analysis and statistical methods, as described.
 While these techniques are commonly known in the art, the invention
 encompasses their use in the detection of a mutation in the IPF-1 gene for
 clinical diagnostic applications.
 EXAMPLE 1
 Genomic cloning of human IPF1.
 To examine the patient's IPF1 gene for the presence of mutations, it was
 necessary to determine the nucleotide sequence of the normal human gene.
 500,000 recombinant phage from a human genomic library (HUVEC, .lambda.
 DASH II, Stratagene, Inc., La Jolla, Calif.) were screened using a .sup.32
 P-labeled PCR-generated probe corresponding to the 5' end of the open
 reading frame in the mouse Ipf1 cDNA (FIG. 1; hatched box) using standard
 methods (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual,
 Cold Spring Harbor Laboratory Press, New York). This probe was chosen
 because of the high degree of sequence similarity between rat and mouse
 IDX-1/IPF-1-encoding cDNAs in this region and to avoid the homeodomain
 that is highly conserved among all members of the homeodomain protein
 family. Southern blot analysis of human genomic DNA hybridized to this
 probe revealed a single band, indicating the likelihood that a single
 human gene contains sequences homologous to this probe. Four rounds of
 screening resulted in two plaque-purified clones, G3 and G5, from which
 phage DNA was prepared. Phage inserts were mapped by Southern analysis of
 partial restriction enzyme digests with .sup.32 P-labeled oligonucleotide
 probes complementary to phage sequences adjacent to the cloning site of
 .lambda. DASH II. FIG. 1A presents a restriction map of overlapping
 genomic phage inserts, G3 and G5, showing the restriction sites of NotI
 (N) and BamHI (B). The library screening probe is indicated by a hatched
 rectangle. The putative positions of the exons are denoted E1 and E2. The
 structure of the mRNA is shown (open box), where UTR represents the
 untranslated region, TAD the transactivation domain, P the pentapeptide
 PBX interaction motif and HD the homeodomain.
 The two human genomic phage clones representing overlapping segments of the
 human IPF1 gene isolated in this screen were partially sequenced after
 mapping. The contig consists of 15 kb and encompasses the entire coding
 region, which consists of 2 exons based on a comparison with the human
 cDNA sequence (Stoffel et al., 1995, Genomics, 28: 125-126; Inoue et al.,
 1996, Gene. Diabetes, 45: 789-794), as shown in FIG. 1A. A schematic
 representation of the IPF1 gene that indicates the locations of three
 blocks of genomic sequence, denoted contig 1 [SEQ ID NO: 22], contig 2
 [SEQ ID NO: 23] and contig 3 [SEQ ID NO: 24], is presented in FIG. 9. The
 coding sequences of the human and mouse genes exhibit 100% amino acid
 identity in the homeodomain and high (86%) amino acid homology in the
 flanking domains.
 Analysis of patient DNA
 Genomic DNA was isolated from peripheral blood samples of the patient,
 parents and normal control subjects by standard methods (Sambrook et al.,
 1989, supra). The nucleotide sequence of both exons was determined after
 PCR amplification of the patient's genomic DNA and subcloning of the
 amplified fragments. PCR amplification of exon 1 was performed in 2
 sequential rounds using nested primers.
 Round 1 primers were:
 PCR4: 5' GGAATTCGGCTGTGGTTCCCTCT 3' [SEQ ID NO: 4]
 and
 S16: 5' CAGAGAGAAGGCTCCTG 3'. [SEQ ID NO: 5]
 Round 2 primers were:
 S17b: 5' AGCGAGCAGGGGTGGCG 3' [SEQ ID NO: 6]
 and
 S18: 5' GGGACGCTTGGAGGTAA 3'. [SEQ ID NO: 7]
 Each round consisted of 26 cycles with denaturation at 97.5.degree. C. for
 15 sec, annealing at 60.degree. C. for 90 sec, and extension at 72.degree.
 C. for 2 min. Where indicated, the resultant fragment (610 bp) was
 subcloned (TA cloning vector PCRII; Invitrogen) and sequenced (Sequenase
 2.0; United States Biochemicals, Cleveland, Ohio). Exon 2 screening was
 performed in 3 overlapping PCR fragments. The sequences of the primer
 pairs employed were as follows:
 (A) PCR2: 5' CGGGATCCGCCGAGCTTCTTGTC 3' and [SEQ ID NO: 8]
 PCR3: 5' GGATTCTGGGGCTTGGTGGCTC 3' [SEQ ID NO: 9]
 (T.sub.ann = 60.degree. C.)
 (B) S1: 5' CGCCTACGCTGCGGAGC 3' and [SEQ ID NO: 10]
 S14: 5' AGAAGCTCCTCGCCGGAG 3' [SEQ ID NO: 11]
 (T.sub.ann = 37.degree. C.)
 (C) S12: 5' AGGAGGAGGACAAGAAGC 3' and [SEQ ID NO: 12]
 PCR7: 5' CGGATCCTAGGGCCTCTGCTCC 3' [SEQ ID NO: 13]
 (T.sub.ann = 37.degree. C.)
 A deletion of a single cytosine was found in codon 63 of exon 1 resulting
 in a frame shift of translation that terminates 59 codons downstream; the
 DNA sequences of an exon 1 PCR subclone from a control subject (left) and
 from the affected patient (right) in this region are shown in FIG. 1B,
 where the deleted cytosine in the patient's DNA is indicated with an
 arrow. In order to confirm that the IPF1.DELTA.C mutation leads to a loss
 of IPF-1 function and, consequently, the patient's pancreatic deficiency,
 it was necessary to analyze the mutant IPF-1 protein.
 Analysis of the IPF1 mutant protein.
 The deletion of a cytosine in codon 63 of human IPF1 results in a
 frameshift beginning at the C-terminal border of the transactivation
 domain of IPF-1. A truncated protein comprising 121 amino acids, with an
 approximate molecular weight of 13.2 kDa, is predicted. In FIG. 2A, the
 nucleotide sequence beginning at codon 61 through the premature stop codon
 is shown [SEQ ID NO: 1], and the deleted cytosine in codon 63 is
 underlined. The amino acid sequence of the wild type reading frame [SEQ ID
 NO: 2] is shown above the nucleotide sequence, and the frame shifted
 reading frame is shown below [SEQ ID NO: 3]. The premature stop codon
 overlapping codon 122 is indicated by an asterisk (*). FIG. 2B diagrams
 the mutant protein. The positions of the transactivation (TA) and DNA
 binding domains (homeodomain, HD) are indicated. The novel sequence
 resulting from the frame shift is indicated in the hatched box. The
 conceptualized protein terminates amino-proximal to the essential DNA
 binding domain, so is missing the homeodomain as well as the FPWMK motif
 required for the interaction with another homeoprotein, PBX (Peers et al.,
 1995, Mol. Cell. Biol. 15: 7091-7097), but retains the transactivation
 domain required for synergistic interaction with transcription factor E47
 in the transcriptional regulation of the insulin gene (Peers et al., 1994,
 supra). It was predicted that the truncated protein would not be
 transported to the nucleus, because the nuclear localization signal
 resides in the homeodomain (Lu et al., 1996, Endocrinology, 137:
 2959-2967), and so would not interfere with normal IPF-1 function.
 To determine directly whether the conceptual translational reading frame
 imposed by the cytosine deletion in codon 63 results in termination after
 59 additional codons, expression plasmids that contained the wild-type
 sequence and the mutation were prepared and transfected into Cos-1 cells,
 and their expressed protein products analyzed. Since a full-length human
 IPF1 cDNA was not available, a fusion Idx1/IPF1 expression construct was
 created in which the unique conserved AflIII site was used to fuse exon 1
 from human IPF1 in frame to the remainder of exon 1 and exon 2 of rat Idx1
 cDNA (Miller et al., 1994, supra). The wild-type human IPF1 portion was
 the 0.5 kb EcoRI-AflIII fragment from a fully sequenced TA subclone of a
 nonaffected individual, while the mutation-containing first exon fragment
 was taken from a sequenced TA subclone from the proband. Thus, the 5' end
 of the open reading frame including the mutation, the ensuing 59 codons
 and the premature stop codon are all derived from human IPF1. The rat
 derived portion was the 0.9 kb AflIII-BamHI fragment of rat Idx1 pBJ5 (Lu
 et al., 1996, supra). These fragments were ligated into the EcoRI and
 BamHI double-digested eukaryotic expression vector pcmv5 (Chen et al.,
 1991, Cell 66: 327-334). The amino-terminal IDX-1/IPF-1 antiserum
 (.alpha.251) was a rabbit polyclonal antiserum raised against the the
 first 12 amino acids of rat IDX-1 (100% conserved between rat and human).
 Two carboxy-terminal antisera were utilized, Hm66 (.alpha.66) directed
 against GST IDX-1 (164-283) which recognizes predominantly homeodomain
 sequences and .alpha.253, directed against the carboxy-terminal 12 amino
 acids of IDX-1/IPF-1. Transfections into Cos-1 cells were performed using
 diethylaminoethyl-dextran and a brief dimethylsulfoxide shock (Ausubel et
 al., 1995, Current Protocols in Molecular Biology, John Wiley and Sons,
 New York).
 Western blot analysis was performed as follows: Whole cell lysates were
 prepared from transfected Cos-1 cells by lysing freshly isolated cells in
 SDS sample buffer. Lysates were sonicated, then cleared by centrifugation
 (10,000.times.G, 4.degree. C.). Nuclear extracts were prepared by the
 method of Dignam et al. (1983, Nucleic Acids Res., 11: 1475-1489).
 Extracts and lysates were fractionated on SDS-polyacrylamide gels and
 electroblotted onto nitrocellulose membranes (MSI; Westborough, Mass.).
 The membranes were incubated with 1:20,000 dilutions of primary antisera
 and, subsequently, with an alkaline phosphatase-conjugated goat
 anti-rabbit secondary antibody (BioRad Laboratories; Richmond, Calif.).
 Immunoreactive proteins were visualized using the ECL chemiluminescent
 detection system (Amersham, Inc.; Arlington Heights, Ill.), as shown in
 FIG. 2C. Results from whole cell extracts are presented (WC Ext; lanes 1,
 3, 5 and 7) as are those of nuclear extracts (Nuc Ext; lanes 2, 4, 6 and
 8) prepared from Cos-1 cells transfected with pcmv5 (Empty Vec; lanes 1
 and 2), rat IDX-1pBJ5 (rWT; lanes 3 and 4), wild-type human IPF-1/rat
 IDX-1 (h/rWT; lanes 5 and 6) and .DELTA.C mutant human IPF-1/rat IDX-1
 (h/rMUT; lanes 7 and 8). All are probed with .alpha.251 against the amino
 terminus of IDX-1/IPF-1. Whole cell extracts from Cos-1 cells transfected
 with pcmv5 (lane 9), rat IDX-1pBJ5 (lane 10), wild type human IPF-1/rat
 IDX-1 (lane 11) and .DELTA.C mutant human IPF-1/rat IDX-1 (lane 12) are
 probed with .alpha.66 against carboxy-terminal IDX-1/IPF-1 sequences.
 For immunoprecipitations, cells were labeled with .sup.35 S
 methionine/cysteine (Trans .sup.35 S-label, ICN) for 1 hour prior to
 harvesting. Analysis of whole cell lysates was carried out as previously
 described (Ron and Habener, 1992, Genes Dev. 6: 439-453) using the amino-
 and carboxy-terminal antisera described above. FIG. 2D depicts
 immunoprecipitation of .sup.35 S-labeled proteins from transfected Cos-1
 cells with pre-immune antiserum .alpha.PI (lanes 1, 3, 5 and 7) and
 .alpha.251 (lanes 2, 4, 6 and 8). Immunoprecipitates are from cells
 transfected with pcmv5 (Empty Vec; lanes 1 and 2), rat IDX-1pBJ5 (lane 3
 and 4), wild type human IPF-1/rat IDX-1 (lane 5 and 6) and .DELTA.C mutant
 human IPF-1/rat IDX-1 (lane 7 and 8). .sup.35 S-labeled proteins from
 whole cell extracts of transfected Cos-1 cells (pcmv5, lane 9; rat
 IDX-1pBJ5, lane 10; wild-type human IPF-1/rat IDX-1, lane 11; .DELTA.C
 mutant human IPF-1/rat IDX-1, lane 12) using antiserum .alpha.253 were
 also examined. The truncated protein is indicated by an arrow. Asterisks
 (*) denote non-specific cross-reacting proteins endogenous to Cos-1 cells.
 Transfected Cos-1 cells were examined by immunocytochemistry using the
 amino-terminal antiserum (.alpha.251) as follows: Cos-1 cells grown on
 chamber slides (Falcon; Oxnard, Calif.) were fixed with 4%
 paraformaldehyde in phosphate-buffered saline, permeablized in acetone at
 -20.degree. C. and incubated overnight in .alpha.251. In order to
 visualize the antibody/IPF-1 complex, slides were incubated with a
 biotinylated secondary antibody, followed by an avidin-conjugated
 horseradish peroxidase complex (Vectastain ABC System, Vector
 Laboratories; Burlingame, Calif.). Immunocytochemistry of transfected
 Cos-1 cells stained with pre-immune serum (top) or .alpha.251 (N-terminal)
 (wild-type IDX-1I/IPF-1, center and mutant IDX-1/IPF-1, bottom) is shown
 in FIG. 2E. Examples of nuclei (Nu) and cytoplasm (Cy) are indicated
 (arrows).
 Antiserum .alpha.251 detected proteins of 42-43 kDa by Western blot
 analysis (FIG. 2C, lanes 3 and 5) and by immunoprecipitation (FIG. 2D,
 lanes 4 and 6) in Cos-1 cells transfected with wild-type IDX-1/IPF-1
 plasmids. These proteins are also detected by a carboxy terminal specific
 antiserum (FIG. 2C, lanes 10 and 11) and are present in nuclear extracts
 (FIG. 2C, lanes 4 and 6). In contrast, Cos-1 cells transfected with an
 IDX-1/IPF-1 expression plasmid harboring the cytosine deletion in codon 63
 expressed only a 16 kDa protein (FIG. 2C, lane7 and FIG. 2D, lane 8). This
 protein was not detected in nuclear extracts (FIG. 2C, lane 8) and could
 not be visualized with a C-terminal specific antiserum (FIG. 2C, lane 12
 and FIG. 2D, lane 12). The predicted cytoplasmic localization of the
 truncated protein was confirmed by immunocytochemistry of transfected
 Cos-1 cells using the amino-terminal antiserum (FIG. 2E). Expression of
 wild-type IDX-1/IPF-1 was entirely nuclear (center panel), while mutant
 IDX-1/IPF-1 had a striking distribution throughout the cytoplasm (bottom
 panel). By electrophoretic mobility shift assays, transfected cell nuclear
 extracts showed the expected binding to known IDX-1/IPF-1 binding sites
 for wild type IDX-1I/IPF-1 proteins but failed to show specific binding in
 nuclear extracts from mutant Idx1/IPF1-transfected cells (data not shown).
 These data indicate that a truncated protein lacking the homeodomain (and
 nuclear localization signal) is produced as a result of the mutation.
 The SMS-TAAT1 promoter element [SEQ ID NO: 18] of the somatostatin gene is
 an IPF-1 target sequence. Other such sequences are the SMS-TAAT2 [SEQ ID
 NO: 19], SMS-UE-B [SEQ ID NO: 20] (Vallejo et al., 1992, J. Biol. Chem.
 267: 12868-12876) and SMS-PS [SEQ ID NO: 21] regulatory elements of the
 rat somatostatin gene and the FAR-FLAT (INS1-FLAT) [SEQ ID NO: 17]
 regulatory element of the rat insulin 1 gene (German et al., 1992, Mol.
 Cell. Biol., 12: 1777). To confirm the supposition that the truncated
 mutant protein is unable to transactivate, RIN5AH insulinoma cells were
 cotransfected by DEAE-dextran in suspension with the SMS-TAAT1-65SMS CAT
 reporter, in which the TAAT1 element drives expression of the bacterial
 chloramphenicol acetyl-transferase gene, and either vector alone (basal),
 wild-type (WT) IPF-1 or mutant (MUT) Idx1/IPF1 expression plasmid (FIG.
 3). Only the wild-type fusion protein directed transcription of the
 CATgene, as indicated by CAT enzymatic activity measured as percent of
 conversion (acetylation) of .sup.14 C-labeled chloramphenicol.
 Together, these findings establish that the IPF1.DELTA.C mutation results
 in the production of a truncated protein that has undergone the loss of a
 biological function and is, therefore, an inactivating mutation at that
 genetic locus.
 EXAMPLE 2
 Allele-specific hybridization analysis of IPF1.
 The cytosine deletion in codon 63 of the IPF1 gene of the apancreatic
 patient described in the first example was observed in 100% of PCR
 subclones of exon 1 (Table 1) suggesting homozygosity. Evidence for this
 deletion was obtained using 4 different sequencing primers which revealed
 the point deletion on both DNA strands of the PCR subclones, confirming
 that it was not a cloning artifact. No mutations were detected in exon 2.
 The patient has no siblings; however, analysis of the parents' genomic DNA
 indicates that each parent is heterozygous for the same cytosine deletion
 in codon 63. Approximately half of the exon 1 PCR subclones from each
 parent possess the point deletion (Table 1).
 TABLE 1
 # Clones
 Subject # PCR subclones analyzed with DC in codon 63 (%)
 Patient 16 16 (100)
 Father 13 8 (61)
 Mother 12 6 (50)
 Normal Control 14 0 (0)
 (Number and % of exon 1 PCR subclones analyzed possessing the point
 deletion for the patient, both parents and a normal control).
 While the results such as are presented in Table 1 are valid, an assay
 which requires sequencing of multiple PCR clones derived from each patient
 in order to determine whether they are homozygous or heterozygous for a
 mutation in IPF1 is of limited clinical utility, as the procedure is
 time-consuming and expensive; this is particularly true when pedigree
 analysis is undertaken, as numerous subjects must be screened. Since no
 restriction enzyme site was gained or lost as a result of this point
 deletion, conditions were optimized for allele-specific hybridization to
 confirm the homozygous versus heterozygous presence of the mutation in the
 patient and her parents. The conditions were as follows:
 Equal amounts of DNA were loaded in each lane of an agarose gel, verified
 by quantitation of ethidium bromide fluorescence prior to Southern
 transfer. Wild-type (5' ATGTCCGGGGGGCTGCC 3') [SEQ ID NO: 14] and
 mutation-specific (5' CAGGGCAGCCCCCGGAC 3') [SEQ ID NO: 15]
 oligonucleotides were end-labeled with T4 polynucleotide kinase and
 .lambda..sup.32 P-ATP. Hybridizations were performed with
 2-3.times.10.sup.6 cpm/ml of labeled oligonucleotide in 6.times.SSC,
 5.times.Denhardt's solution, 1% SDS, 50 mM sodium phosphate, pH 6.8 and
 100 .mu.g/ml denatured salmon sperm DNA at 37.degree. C. for 6-15 hours.
 Washes were: 4.times.10 min in 5.times.SSC, 0.1% SDS at room temperature,
 followed by 1.times.10 min in 5.times.SSC, 0.1% SDS at 37.degree. C.,
 2.times.30 min in 3M tetramethylammonium chloride, 50 mM Tris, pH 8.0,
 0.2% SDS at 52.degree. C. followed by a brief rinse in 2.times.SSC, 0.1%
 SDS at room temperature (Wood et al., 1985, Proc. Natl. Acad. Sci. USA,
 82: 1585-1588).
 Both parents exhibited an intermediate intensity of hybridization to the
 wild-type sequence and mutation-specific oligonucleotides, supporting
 heterozygote status. The affected patient exhibited no hybridization with
 the wild-type oligonucleotide, while the normal control subject had no
 hybridization with the mutation specific oligonucleotide. These results
 are summarized in FIG. 1C, where Southern blots used for allele-specific
 hybridization, performed to confirm the mutation status in the patient
 (PA), both parents (F=father, M=mother) and a normal control (N) are
 shown, along with graphs below each blot that represent densitometry of
 autoradiograms and/or quantitation of phosphorimager scans. The pedigree
 is depicted above the results. In the case of the wild-type
 oligonucleotide probe (n=2), the patient's density of hybridization was
 set to 0.0 and the normal to 1.0 (based on sequencing of PCR subclones;
 see Table 1) after an initial background correction. Conversely, for the
 mutation-specific oligonucleotide (n=4), the patient's density of
 hybridization was set to 1.0 and that of the normal control to 0.0.
 Similar results were obtained when the higher fidelity Pfu DNA polymerase
 was employed in the PCR reactions. Collectively, these data indicate that
 the parents are heterozygous, while the proband is homozygous, for the
 cytosine deletion in codon 63.
 EXAMPLE 3
 Single-strand conformation polymorphism analysis.
 One approach to detecting multiple allelic isoforms of a gene resulting
 from single base mutations in an organism or plurality of organisms is
 single strand conformation polymorphism (SSCP) (Glavac et al., 1993, Hum.
 Mut., 2:404; Sheffield et al., 1993, Genomics, 16:325). SSCP is a simple
 and effective technique for the detection of single base mutations. This
 technique, with which one can simultaneously screen a patient for the
 presence and allelic copy number of a mutation in IPF1, is based on the
 principle that single-stranded DNA molecules take on unique sequence-based
 secondary structures (conformers) under nondenaturing conditions.
 Molecules differing by as little as a single base substitution may form
 different conformers which assume different electrophoretic mobility
 profiles in a nondenaturing polyacrylamide gel. The genomic region to be
 screened for sequence polymorphisms is first amplified by PCR in each
 sample to be compared. Single-base substitutions as well as short
 insertions or deletions lying within the probe region (between the PCR
 primers) can be detected efficiently. Although the nature of a given
 mutation is not revealed by this technique, the presence of a mutation
 within the amplified region is made apparent, allowing for further
 molecular analysis. This technique has proven useful for detection of
 multiple mutations and polymorphisms. SSCP sensitivity varies dramatically
 with the size of the DNA fragment being analyzed. The optimal size
 fragment for sensitive detection by SSCP is approximately 150-300 bp.
 Typically, electrophoresis of single-stranded DNA is performed under
 denaturing conditions that maintain the single strandedness of the
 molecules. Common denaturants are urea, formamide and sodium hydroxide.
 The SSCP gel is unconventional in that single-stranded DNA is loaded on
 gel that lacks a denaturant. Small molecules would ordinarily pass through
 the pores of the gel matrix more easily than large molecules and migrate
 faster. In a denaturing gel, intramolecular interactions occur, and
 single-stranded DNA is able partially to self-anneal; therefore, its
 mobility is governed by both size and tertiary structure (conformation).
 The technique is performed as follows:
 One or more test nucleic acid samples are prepared for PCR amplification.
 Oligonucleotide primers are synthesized by standard methods. Nested PCR
 amplification of the first exon of IPF1 is performed as described above,
 using primers PCR4 [SEQ ID NO: 4] and S16 [SEQ ID NO: 5] in the first
 round and S17b [SEQ ID NO: 6], except that in the second round,
 amplification and radiolabeling are performed concurrently. A typical
 deoxynucleotide mix would include 0.2 mM of non-radioactive dGTP, dATP,
 dTTP, 0.02 mM of non-radioactive dCTP and 0.05 ml of [.alpha.-.sup.33 P]
 dCTP (1,000-3,000 Ci/mmol; 10 mCi/ml).
 SSCP analysis is performed as follows. Ten .mu.l of formamide dye (95%
 formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) are
 added to 10 .mu.l of radioactive PCR products. The reactions are denatured
 at 100.degree. C. for 5 min, then quenched on ice. Two .mu.l are loaded
 onto a gel containing 8% acrylamide:bisacrylamide (37.5:1), 0.5.times.TBE
 (45 mM Tris-borate, 1 mM EDTA) and 5% glycerol and electrophoresed in
 0.5.times.TBE at 25W for 8 hours at 4.degree. C. Dried gels are exposed to
 X-OMAT ARfilm (Kodak) and the autoradiographs are scored for the presence
 of bands that do not co-migrate with those generated by a wild-type
 control nucleic acid sample, which is run side-by-side with test samples;
 a sample with an aberrant electrophoretic profile contains a mutation in
 the region of interest. SSCP may be optimized, as desired (Glavac et al.,
 1993, supra).
 It is also advantageous to examine nucleic acid samples for mutations in
 IPF1 using fluorescent SSCP (fSSCP) assays (Makino et al., 1992, PCR
 Methods Appl. 2:10; Ellison et al., 1993, Biotechniques, 15:684). In the
 second round of nested PCR amplification, fluorescently-labeled
 deoxynucleotides can be incorporated and the reaction products visualized
 and analyzed using an ABI fluorescent DNA sequencing machine. Four
 distinct fluorochromes now available can be used for different primer
 pairs, and by differentially labeling the test and control PCR products,
 the two samples can be loaded into the same lane of a gel for direct
 comparison, eliminating lane-to-lane variations in gel resistance and,
 consequently, the rate of sample migration. A major advantage of fSSCP
 over SSCP is that the latter requires handling of radioactive materials
 whereas fSSCP does not. Data collection is automated, and data analysis
 programs can be used to identify aberrantly-migrating samples, whereas
 SSCP evaluation involves visual examination, and correction for
 lane-to-lane variations in electrophoretic conditions is not possible.
 fSSCP analysis is performed as follows.
 Amplifications are performed as above, except that a standard
 deoxynucleotide mix (0.2 mM of dGTP, dATP, dTTP, dCTP) is employed and
 each second-round reaction contains a primer labeled with one of the
 fluorochromes HEX, FAM, TET or JOE. Two .mu.l of fluorescent PCR products
 are added to 3 ul formamide dye (95% formamide, 20 mM EDTA, 0.05%
 bromophenol blue, 0.05% xylene cyanol), denatured at 100.degree. C. for 5
 min., then chilled. As an internal standard, 0.5-1 ml of Genescan.TM. 1500
 size markers is added, then 2 .mu.l of the mix are loaded onto a gel
 containing 8% or 10% acrylamide:bisacrylamide (37.5:1), 0.5.times.TBE (45
 mM Tris-borate, 1 mM EDTA) and 5% glycerol. Electrophoresis is performed
 in 0.5.times.TBE at 4.degree. to 10.degree. C. at 2500-3500 volts for 4 to
 10 hours on an ABI 377 DNA sequencing machine. Data is automatically
 recorded and analyzed with Genescan.TM. and Genotype analysis software
 (ABI). As with radioactive SSCP, peaks of fluroescence that vary in
 electrophoretic mobility relative to those of control samples indicate the
 presence of a mutation.
 In the case either of SSCP or fSSCP, the fragment which shows the aberrant
 migration is amplified again from the mutated nucleic acid sample using
 unlabeled primers and sequenced according to standard molecular methods.
 EXAMPLE 4
 The present invention can be used for screening a plurality of individuals
 for defects in the IPF1 gene. In this manner, the invention is useful both
 in population genetics research and in linkage analysis of IPF1 mutations
 and pancreatic disease in a candidate pedigree. It was decided to
 determine the frequency of occurrence of the IPF1.DELTA.C mutation in a
 population of unaffected subjects. We used the mutation-specific
 oligonucleotide under the hybridization conditions employed in Example 2
 to screen genomic DNA from 92 normal control subjects, representing 184
 independent chromosomes, for the presence of the cytosine deletion. This
 mutation was not detected in any of the control subjects, decreasing the
 likelihood that the point deletion is simply a DNA sequence polymorphism.
 Given the rarity of the IPF1.DELTA.C allele and the high incidence of
 early-onset diabetes in both branches of the proband's family, it was
 decided to determine whether or not the risk of diabetes is linked to the
 .DELTA.C mutation in this pedigree.
 Genomic DNA was prepared from peripheral blood samples of 27 members of
 this pedigree, and Exon 1 of the IPF1 gene was amplified using nested
 primers PCR4 [SEQ ID NO: 4] and S16 [SEQ ID NO: 5] in the first round and
 S17b [SEQ ID NO: 6] and S18 [SEQ ID NO: 7] in the second, according to the
 protocol described in Example 1, and allele-specific hybridization was
 performed on the PCR product using primers of the wild-type [SEQ ID NO:
 14] and mutant [SEQ ID NO: 15] IPF1 sequences, as described above in
 Example 2. As in previously reported MODY pedigrees, the disease follows
 an autosomal dominant mode of transmission with age-dependent penetrance
 in six generations. The average age of onset is 35 years (range 17-67) and
 6 of 8 affected heterozygotes are treated with diet and/or oral
 hypoglycemic agents.
 Linkage results were analyzed with MLINK and ILINK programs from the
 LINKAGE package version 5.1, under a model of autosomal dominant
 inheritance. A disease gene frequency of 0.01 and equal male to female
 recombination was assumed. Marker allele frequencies were estimated from
 the genotypes observed within the MODY4 pedigree. For the IPF1.DELTA.C
 allele, the frequency was set at 0.001. Two-point LOD scores were
 insensitive to modifications of disease gene frequencies to 0.01-0.0001,
 and to setting marker allele frequencies at 1/n, where n is the total
 number of alleles for a given marker. To account for the age-dependent
 penetrance of MODY and the existence of phenocopies caused by sporadic
 late-onset diabetes, LOD scores were calculated using 4 liability classes
 stratified by age of onset for affected subjects or age of last clinical
 assessment for unaffected subjects): I, &lt;15; II, 15-30; III, 30-60; IV,
 &gt;60 years. Penetrance was set at 0, 0.5, 1, and 1 for susceptible
 genotypes, on the basis of the mean age of diagnosis in affected
 individuals of this pedigree (35 years). LOD score calculations remained
 &gt;3 with variations of penetrance in class II between values 0.2-0.8.
 Penetrance for nonsusceptible homozygotes was set at 0.001, 0.001, 0.005,
 0.05. These values are based on prevalence rates of diabetes in U.S.
 Caucasians (National Diabetes Data Group in Diabetes in America; National
 Institutes of Health, Bethesda, 1995). Similar assumptions have been used
 in previous linkage studies for MODY1, 2 and 3 (Froguel et al., 1992,
 supra; Hattersley et al., 1992, Lancet, 339: 1307-1310; Zhang et al.,
 1995, Diabetologia, 38: 1055-1060; Bowden et al., 1992, Am.J.Hum.Genet.,
 50: 607-618; Bell et al., 1991, Proc. Natl. Acad. Sci. USA 88: 1484-1488;
 Menzel et al., 1995, Diabetes, 44: 1408-1413).
 The results are presented in FIG. 4. The subjects are identified by
 generation number (along left margin) and individual numbers within each
 generation (upper left of symbols). Diabetic phenotype is indicated by
 filled symbols. The shaded symbol indicates impaired glucose tolerance.
 Genotype is indicated below the identification number (N=normal sequence
 allele, M=allele with IPF1.DELTA.C mutation). The sixth generation
 offspring is homozygous for IPF1.DELTA.C and has pancreatic agenesis and
 diabetes as well as exocrine pancreatic efficiency (indicated by a hatched
 circle). Age at diagnosis of diabetes or age of ascertainment (if
 unaffected) is indicated below the genotype. Treatment modality is
 indicated below the age (OHA=oral hypoglycemic agent, Ins=insulin).
 The IPF1.DELTA.C mutation segregated with early-onset diabetes in both
 branches of the pedigree, and two-point LOD scores provided significant
 evidence for linkage to this locus (Z=3.43, .theta.=0). Although one
 consanguinous loop is observed in this pedigree, subject I-1 is not
 diabetic and carries two normal alleles, suggesting that 1-2 transmitted
 the mutation and may thus also be genetically related to I-1. Consistent
 with this circumstance, both I-2 and her mother were diabetic. Subject
 III-5 had glucose intolerance and carried the IPF1.DELTA.C mutation. Three
 unaffected individuals heterozygous for IPF1.DELTA.C are under age 30, but
 are well within the age range at which diabetes develops in this family.
 Subject III-2 is diabetic but does not carry the mutant allele. Because
 she has late-onset type II diabetes (age of diagnosis 67 years), she is
 likely to represent a phenocopy. Sporadic late-onset diabetes has been
 observed in all forms of MODY where the genetic defect is known (Froguel
 et al., 1992, supra; Hattersley et al., 1992, supra; Yamagata et al.,
 1996, Nature, 384: 455-458; Yamagata et al., 1996, Nature, 384: 458-460;
 Velho et al., 1997, Diabetologia, 40: 217-224).
 EXAMPLE 5
 To assess for linkage to MODY1, 2 and 3, amplification of simple sequence
 repeat polymorphisms of markers closely linked to them was performed using
 standard PCR conditions. These markers included ADAPR (MODY1), GCK1 and
 GCK2 (MODY2) and D12S76 (MODY3). One of each pair of primers was
 end-labeled with .lambda..sup.32 P-ATP and T4 polynucleotide kinase
 (Promega Corp.; Madison, Wis.) and 0.5 .mu.l of labeled primer was
 included in each 15 .mu.l PCR reaction. The amplified products were
 resolved on a 6% denaturing sequencing gel, the gel was dried and exposed
 to film. The observed alleles were independently scored by two
 individuals.
 When family members were genotyped with these markers, all resulted in
 negative LOD scores at low recombination values (Table 2). These
 observations provide evidence that diabetes in this family is not linked
 to any of the three previously characterized MODY loci. Based on the
 clinical phenotype, the mode of transmission, and the linkage analysis
 results, it is apparent that IPF1 represents a new genetic locus of
 diabetes, MODY4.
 EXAMPLE 6
 Interestingly, two of the eight reported cases of pancreatic agenesis,
 referenced above, involved siblings (Sherwood et al., 1995, supra), while
 another involved documented consanguinity (Mehes and Vamosk, 1976, supra).
 These two reports and our data indicate that some cases of pancreatic
 agenesis are transmitted in an autosomal recessive manner. Given the
 rarity of pancreatic agenesis, an extended haplotype analysis using two
 closely linked microsatellite markers (Ipf1CA2 and cdx3GA1; Inoue et al.,
 1996, Gene. Diabetes, 45: 789-794) was performed in the proband and her
 parents, as well as several unrelated controls. Amplimers and PCR
 conditions for simple sequence repeat polymorphisms, Ipf1CA2 and cdx3GA1,
 were as described (Inoue et al., 1996, supra). Diagrammatic representation
 of scored alleles is shown in in the upper portion of FIG. 5, while the
 lower portion presents an autoradiogram of end-labeled PCR products
 resolved on a 5% denaturing sequencing gel (PA, proband; +, wild-type IPF1
 allele; C, allele possessing the cytosine deletion in codon 63). For
 Ipf1CA2, 3 distinct alleles were resolved in the proband, parents and four
 normal controls. For cdx3GA1 , 5 alleles were observed.
 This analysis was consistent with homozygosity by descent of the
 chromosomal segment containing IPF1-.DELTA.C. Thus, as expected for a rare
 allele ocurring in the homozygous state, both of the proband's
 IPF1-.DELTA.C alleles are likely to have been derived from a single common
 ancestor.
 EXAMPLE 7
 It is clear from the experiments described in Example 1 that the
 IPF1.DELTA.C mutation results in the premature termination of the
 translation of IPF-1 and that the truncated product lacks the C-terminal
 domain of the protein consisting of the homeodomain that contains the
 DNA-binding and nuclear translocation functions. Subsequent to those
 experiments, we observed that .alpha.-253, the antibody specific for the
 detection of the C-terminus of IPF-1, detects a protein encoded by the
 mutated IPF-1 mRNA that translocates to the nucleus. FIG. 6A shows
 immunocytochemistry of Cos-1 cells transfected with wild-type (left hand
 panels) or mutant IPF-1 cDNA (right hand panels) stained with N-terminal
 (.alpha.N; upper panels) and C-terminal (.alpha.C; lower panels) specific
 antisera .alpha.251 and .alpha.253, respectively. Examples of stained
 nuclei (Nu) and cytoplasm (Cyt) are indicated. It has been shown that
 IPF-1 is the predominant transcription factor binding to the rat insulin-1
 gene glucose-responsive FAR-FLAT enhancer element [SEQ ID NO: 17] (Peers
 et al., 1994, supra). We performed electrophoretic mobility shift assays
 on this element according to the method of Miller et al. (1994, supra),
 using nuclear extracts prepared from baby hamster kidney (BHK) cells that
 were lipofectin transfected with increasing amounts (1.25-12.5 mg) of
 expression vectors containing either wild-type IDX1/IPF1 or mutant
 IDX1/IPF1 cDNA harboring the IPF1.DELTA.C mutation, the construction of
 which is described above. The mobility shift assay was performed using
 labeled, double-stranded FAR FLAT element of the rat insulin I promoter as
 probe. We observed that a translational product of the mutant cDNA binds
 to this IPF-1-specific element. In FIG. 6B, the specific complex is
 indicated by an arrow.
 These unanticipated findings prompted us to more closely examine the
 nucleotide sequence of exon 1 of IPF-1 for the possibility of an internal
 reinitiation of translation of the mutated IPF-1 mRNA triggered by the
 premature termination of translation. Such occurrences in the translation
 of prematurely translated mRNAs are well recognized and are believed to
 result from the release of ribosomes initiated by 5' upstream AUG start
 codons thus relieving ribosomal elongation occlusion and allowing for
 reinitiation of translation at cryptic internal AUG codons in the mRNA
 (Walker and Habener, 1996, J. Biol. Chem. 271: 20145-20150; Kozak, 1995,
 Proc. Natl. Acad. Sci. USA 92: 2662-2666). Upon examination of the
 nucleotide sequence of the IPF-1 mRNA we found an AUG codon in a favorable
 context for translational initiation (as defined by Kozak, 1989, J. Cell
 Biol., 108: 229-241) at nucleotides 51 to 53 and determined that this AUG
 codon reinitiates translation of the mutant IPF-1 mRNA. Site-directed
 mutagenesis of the out-of-frame AUG beginning at nucleotide 51 of the
 IDX1/IPF1 open reading frame was performed such that A was replaced by G
 at position 51 using the Quik-Change Kit (Stratagene; La Jolla, Calif.).
 This mutation converted the methionine codon to a valine while leaving the
 amino acid coding sequence of the N-terminal truncated translation product
 unchanged; no C-terminal IPF-1 protein is produced in this case. Western
 blot analysis of nuclear extracts prepared from Cos-1 (lanes 1 and 2) and
 BHK (lanes 3 and 4) cells transfected with mutant IPF-1 expression plasmid
 (lanes 1 and 3) and the same plasmid in which the AUG at nucleotides 51-53
 was eliminated by site-directed mutagenesis to a GUG (Val) (lanes 2 and 4)
 is shown in FIG. 6C. The protein band which is lost as a result of the AUG
 mutation is indicated by an arrow, and the position of the 43 kDa
 molecular weight marker is indicated. The larger species observed in the
 Cos-1 extracts is believed to initiate at an upstream site in the
 polylinker of the expression vector. Although translation is out of the
 reading frame for IPF-1, the mutation IPF1.DELTA.C in the mutant IPF1
 allele shifts and reestablishes the correct reading frame for the protein.
 FIG. 7 presents the nucleotide sequence of exon 1 [SEQ ID NO: 1] along
 with the translated regions of the three open reading frames (ORF-1, 2 and
 3) [SEQ ID NO: 2, 3 and 16, respectively] in single-letter amino acid
 code. The position of the IPF1.DELTA.C mutation is indicated by .DELTA.C.
 A schematic depiction of the two mutant IPF-1 isoforms and the open
 reading frames which are joined as a result of the cytosine deletion in
 codon 63 (.DELTA.C) is shown in FIG. 8 (ORF1, open box; ORF2,
 cross-hatched; ORF3, stippled; TA, transactivation domain; DBD,
 DNA-binding domain; HD, homeodomain; NLS, nuclear localization signal).
 The consequence of this translational frame-shift is the synthesis of an
 alternative isoform of IPF-1 that lacks the N-terminal domain critical for
 the transactivation of gene transcription (Lu et al., 1996, supra; Peers
 et al., 1994, supra) but possesses the DNA-binding and nuclear
 translocation signals.
 These findings raised the possibility that the N-terminally truncated,
 internally-translated IPF-1 isoform may serve as a dominant negative
 inhibitor of the wild type protein expressed from the unaffected IPF1
 allele. To test this assumption, RIN5AH insulinoma cells were transfected
 by DEAE-dextran in suspension with expression plasmid containing either
 the mutant or wild-type Idx1/IPF1 construct along with a rat insulin-1
 promoter CAT reporter plasmid. Indeed, the mutant protein is able to
 reduce basal transcription of a rat insulin 1 reporter in RIN5AH cells
 (FIG. 6D).
 USE
 The present invention is of use in the assessment of an individual's risk
 of developing mature onset diabetes of the young 4 (MODY4), which results
 from an inactivating defect in the gene encoding insulin promoter factor-1
 (IPF-1). Such a genetic screen is particularly valuable in evaluating the
 prognosis of an individual in whose family early-onset diabetes type II is
 known to exist.
 The invention can be applied to a plurality of subjects, either unrelated
 individuals or those belonging to an extended family, to establish the
 linkage between pancreatic disease and mutations in IPF1 in a given
 population or pedigree.
 An additional use of the invention is in the field of genetic couseling. It
 is advantageous to know the specific genetic basis for an inherited
 disease, even when risk analysis is not an issue, i.e. when a patient has
 already become symptomatic. In the case of a disorder that is linked to
 defects at multiple genetic loci, assignment of the mutation to a specific
 locus in a given patient can be used when two affected individuals are
 deciding whether or not to have children together. While both might suffer
 from MODY, it is not necessarily true that both would have inactivating
 mutations in IPF1; if not, their child would not be at risk of pancreatic
 agenesis.
 It is further envisioned that the methods of the invention can be applied
 to prenatal screening for pancreatic agenesis in those cases in which both
 of the unborn patient's parents are either known to carry- or are
 suspected of carrying an inactivating mutation in IPF1.
 TABLE 2
 Pairwise LOD scores for linkage between diabetes in the family
 pedigree and markers for the MODY 1, 2, and 3 loci and Pro63fsdelC
 LOD score at recombination fraction (.theta.) of
 Marker 0 0.001 0.01 0.1 0.2 0.4
 ADAPR (MODY1) -7.22 -4.11 -2.87 -0.98 -0.41 -0.06
 GCK1 (MODY2) -4.86 -3.84 -2.68 -1.06 -0.56 -0.11
 GCK2 (MODY2) -2.74 -2.66 -1.97 -0.44 -0.10 0.03
 D12S76 (MODY3) -6.62 -6.53 -5.32 -1.93 -0.98 -0.19
 Pro63fsdelC 3.43 3.42 3.37 2.86 2.19 0.66
 Maximum 2-point LOD scores for each marker are shown in bold.
 SEQUENCE LISTING
 (1) GENERAL INFORMATION:
 (iii) NUMBER OF SEQUENCES: 24
 (2) INFORMATION FOR SEQ ID NO: 1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 400 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: human IPF-1 gene
 (B) LOCATION: exon 1
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
 CCAATCCCGG GCCGCAGCCA TGAACGGCGA GGAGCAGTAC TACGCGGCCA CGCAGCTTTA 60
 CAAGGACCCA TGCGCGTTCC AGCGAGGCCC GGCGCCGGAG TTCAGCGCCA GCCCCCCTGC 120
 GTGCCTGTAC ATGGGCCGCC AGCCCCCGCC GCCGCCGCCG CACCCGTTCC CTGGCGCCCT 180
 GGGCGCGCTG GAGCAGGGCA GCCCCCCGGA CATCTCCCCG TACGAGGTGC CCCCCCTCGC 240
 CGACGACCCC GCGGTGGCGC ACCTTCACCA CCACCTCCCG GCTCAGCTCG CGCTCCCCCA 300
 CCCGCCCGCC GGGCCCTTCC CGGAGGGAGC CGAGCCGGGC GTCCTGGAGG AGCCCAACCG 360
 CGTCCAGCTG CCTTTCCCAT GGATGAAGTC TACCAAAGCT 400
 (2) INFORMATION FOR SEQ ID NO: 2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 124 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: human IPF-1 protein
 (B) LOCATION: amino acids 1 through 124
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
 Met Asn Gly Glu Glu Gln Tyr Tyr Ala Ala Thr Gln Leu Tyr Lys Asp
 1 5 10 15
 Pro Cys Ala Phe Gln Arg Gly Pro Ala Pro Glu Phe Ser Ala Ser Pro
 20 25 30
 Pro Ala Cys Leu Tyr Met Gly Arg Gln Pro Pro Pro Pro Pro Pro His
 35 40 45
 Pro Phe Pro Gly Ala Leu Gly Ala Leu Glu Gln Gly Ser Pro Pro Asp
 50 55 60
 Ile Ser Pro Tyr Glu Val Pro Pro Leu Ala Asp Asp Pro Ala Val Ala
 65 70 75 80
 His Leu His His His Leu Pro Ala Gln Leu Ala Leu Pro His Pro Pro
 85 90 95
 Ala Gly Pro Phe Pro Glu Gly Ala Glu Pro Gly Val Leu Glu Glu Pro
 100 105 110
 Asn Arg Val Gln Leu Pro Phe Pro Trp Met Lys Ser
 115 120
 (2) INFORMATION FOR SEQ ID NO: 3:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 59 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
 Arg Thr Ser Pro Arg Thr Arg Cys Pro Pro Ser Pro Thr Thr Pro Arg
 1 5 10 15
 Trp Arg Thr Phe Thr Thr Thr Ser Arg Leu Ser Ser Arg Ser Pro Thr
 20 25 30
 Arg Pro Pro Gly Pro Ser Arg Arg Glu Pro Ser Arg Ala Ser Trp Arg
 35 40 45
 Ser Pro Thr Ala Ser Ser Cys Leu Ser His Gly
 50 55
 (2) INFORMATION FOR SEQ ID NO: 4:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 23 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer PCR4
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
 GGAATTCGGC TGTGGTTCCC TCT 23
 (2) INFORMATION FOR SEQ ID NO: 5:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer S16
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5
 CAGAGAGAAG GCTCCTG 17
 (2) INFORMATION FOR SEQ ID NO: 6:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer S17b
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
 AGCGAGCAGG GGAGGCG 17
 (2) INFORMATION FOR SEQ ID NO: 7:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer S18
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
 GGGACGCTTG GAGGTAA 17
 (2) INFORMATION FOR SEQ ID NO: 8:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 23 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer PCR2
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
 CGGGATCCGC CGAGCTTCTT GTC 23
 (2) INFORMATION FOR SEQ ID NO: 9:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 22 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer PCR3
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9
 GGATTCTGGG GCTTGGTGGC TC 22
 (2) INFORMATION FOR SEQ ID NO: 10:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer S1
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
 CGCCTACGCT GCGGAGC 17
 (2) INFORMATION FOR SEQ ID NO: 11:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 18 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer S14
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11
 AGAAGCTCCT CGCCGGAG 18
 (2) INFORMATION FOR SEQ ID NO: 12:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 18 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer S12
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12
 AGGAGGAGGA CAAGAAGC 18
 (2) INFORMATION FOR SEQ ID NO: 13:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 22 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: primer PCR7
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
 CGGATCCTAG GGCCTCTGCT CC 22
 (2) INFORMATION FOR SEQ ID NO: 14:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (D) OTHER INFORMATION: Wild-type primer for allele-specific
 hybridization of IPF-1 gene.
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
 ATGTCCGGGG GGCTGCC 17
 (2) INFORMATION FOR SEQ ID NO: 15:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 17 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: other nucleic acid
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (D) OTHER INFORMATION: Mutant primer for allele-specific
 hybridization analysis of IPF-1 gene.
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15
 CAGGGCAGCC CCCGGAC 17
 (2) INFORMATION FOR SEQ ID NO: 16:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 45 amino acids
 (B) TYPE: amino acid
 (D) TOPOLOGY: unknown
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16
 Met Arg Val Pro Ala Arg Pro Gly Ala Gly Val Gln Arg Gln Pro Pro
 1 5 10 15
 Cys Val Pro Val His Gly Pro Pro Ala Pro Ala Ala Ala Ala Ala Pro
 20 25 30
 Val Pro Trp Arg Pro Gly Arg Ala Gly Ala Gly Gln Pro
 35 40 45
 (2) INFORMATION FOR SEQ ID NO: 17:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 32 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: INS-FLAT element.
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17
 GATCCTTCTT AATCTAATTA CCCTAGGTCT AA 32
 (2) INFORMATION FOR SEQ ID NO: 18:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 30 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: SMS-TAAT1 element
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18
 GATCCCTGAT TGCATATTAA TTCTCAGATA 30
 (2) INFORMATION FOR SEQ ID NO: 19:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 30 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: SMS-TAAT2 element
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19
 GATCCGATCT CAGTAATTAA TCATGCACCA 30
 (2) INFORMATION FOR SEQ ID NO: 20:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 37 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: SMS-UE-B element
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20
 GATCCGCGAG GCTAATGGTG CGTAAAAGCA CTGGTGA 37
 (2) INFORMATION FOR SEQ ID NO: 21:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 24 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: SMS-PS element
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21
 GATCCAGGCA AGATTATTTG GTCA 24
 (2) INFORMATION FOR SEQ ID NO: 22:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 4853 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: IPF1 gene, contig 1.
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22
 TCACTGCCAC CCCCNNGTGN TTACTTNGNA CTCCTCAACT AATTGAACAT TTCTGTAAAT 60
 TGGNCTCCAT TTCGATGCTN CATTTGCTNC CATTTANTAT TTGGGCTCGG CCCTNNAGAA 120
 AGAAAAGAGG NTCTCAGAAG AAAGCAACTC CGCCGGGTGG GGGCGCCGAA AAAACCCCGG 180
 CTACCCNCNG TTGGGGGGGA GGAGGAGGGG ATCGCCGGGG GTATCCCATG GNGGCGNNNT 240
 CTCACTAACA CAATTAGTAT TTTCTGCTGC CTGCGACCTA TAGAGGGGCC CCAGGGAGCA 300
 AAGSTCANNC AMCCATTTNT TATTGTTATT ATTATTATNA GATNAAACAA WGTTATCTGG 360
 AATTTTATCA CGTACAGCCG TATAAAAAAC GCATCAAGCG GAACCCTCCG CTTAGTAAGG 420
 TCGTGTGTTG TACTTCCCCG TGGATCTAGC TCCTGGTATC GTAAAATCGC CGAGTCCATT 480
 ACAGGCGAAA TGCGTTTCCG CTCTGAATCC CCGCTGACGC CTGCGCCCCC TCCAAGCCGC 540
 CTCGCCCACA ACAGGCCCGG CAGCGATACC GAGCCATTAG GCGAGCACCT TCCCGAGCCA 600
 TTTAACAGCA GCTCTTATGG ATAAATAAAC AAAAAAGGCT GTAAACCAAT TAAAGTGTGG 660
 ACAGTGAAAA AGTCGTTTAT TAGCTGGACG TCCTGATAGT CCTCGCTGAT ACTGGAGGTC 720
 CTTGCCGGCC CTCTTCCCCC TTTCCCTTCA CAGAGACGCG GTTTACACCC GGGAGAACAC 780
 AGGTTACCTT GTTCTTGGTA AGTGGCTAAG TCTTCCTCGG TGGTTTCAGA GGCATGCTAA 840
 GGGCTGAATT TGGCTTGGTG ATAGGATTTT TAAGCCACTG CATCCCATTA TAAATGAAAA 900
 AAACAGATGA ATCCCACTTA ACTCCAAGGT ATACATCATG AATGCACGTG GAATTTATAT 960
 TCATTTTATA ATTAAATCTC TGTTGAAAAT AAAAATTGTT TCTTGAGTTG GAAAATTCTG 1020
 TAAAGCAAAC GATGTGATGT GTGGAATGAG AATACTTCTA TAATTGATTG CACTTTAGTT 1080
 CATTAGCATT TCCTTCAATG AGCACTAATG CAGGCAGGTT TTCATTTTCC TAAGTCGAAT 1140
 CAATGTTTCC CTCCTGCTGG TTTATGAATA CCCAGAATTG GCCGAGGGCG KKGGCTCACG 1200
 CCTGTAATCC CAGCACTTTG GGAGGCTGAG ACAGGCGGAT CATGAGGTCA GNGAKWTCGA 1260
 GACCATCCTG GCTAACATGA TGAAACCCCG TCTCTACTAA ACCTACAAAA AAATTTAGCC 1320
 GGGCGTGGTG GCGGGCGCCT GTAGTTCCAG CTACTTGGGA GGCTGAGGAA GGAGAATGGC 1380
 GTGAACCCAG GAGGCGGAGC TTGCAGTGAT CCGAGATCGC GCCACTGCCC TCCAGCCTNG 1440
 GAGACAGAGC GAGGCTCCGT CTCAAAAAAT TAAAAATATA TATATATATA TAAATAAATA 1500
 CCCAGAATTT GGTATCCAGG TCTGAGAGGG GCCAGGGAAA CCCAGCTTGG GTTGTGGGGA 1560
 TTGGAGAGAG GAAAGGACCT CAGACTTTGA ATGAANGGTT TTCCAATATT CCTAGGGCTA 1620
 AGCAATCTAG TGAAAATAAG CAGAGAGCTG AAATTATTTT CCAGATGATT CAGTGATCCC 1680
 TGAACTGTAA AGATAAAAGC AGCTTAAAAA CCGAATTGAA TTGGGTAATT GTCTTATTCC 1740
 CTACCTTGTT AGCTCTCTCT GGTGTAAGAG GAGTAGAAGG GCAGGAGGGG GGACTCTGTG 1800
 TGCCCCAGAA CTGTCTGCTG CTTCCTCCAG CCACTGCTCA ACGCTTCCTC CCTGGGAGTG 1860
 GGGCCCCCAG ACACGAGCTC TTTCCACTTT TGGTTTAATC CTTCCGTGGT CCTCAGTTCA 1920
 CTCTGCTGAT GAAAGGAAAA GTATGAAAGG ACTTTCAGGC TCCAAANGAT CTGAGGTCAN 1980
 CTGTTGCYCC CCACTCCTGG TGTTCCCGGT GGCTCACCCC CTACAGCTCT GGACTCACCC 2040
 CTTTGGTTGT CTCATCTCTG GTTCTACCTT CCAGATGTGC AGTCTCCAGA AATCCCTGCT 2100
 GCTTCCCTTC TGAGCTTAGT CCTAGGTAAC CAGCTCCTTG CTTCCACACA GTTCCTTTTC 2160
 TTTGCCTTCC ACCTCCTCCT CCCACCACCA TTTCCTATGA CTAATCCTTT GTTCTCCTAG 2220
 GAGCCTCTCC TTACCTCCAG GTTCCTGCTG CAGCCTAAAG AAGGCTTGAC CCACACTACC 2280
 CTCCAGCTTC CTTCCTCAAC CATCCTACAT TTCACATGGT TTATGCTTTG AAGGACCCAG 2340
 ATTTTCGGTA ACATATACTG ATCAAATGCC TGTAGATCTC TCACTGTGCA CCTGCCTGCA 2400
 CACTCTTTGA GTAACCAAAA CTCATTGGTA GAAATATGCG CTGGCTTTGG CGGGCCATAG 2460
 AAAGGTCAGG AGCTGCATAC ACCTGTCCTC CACATCAGGC TCTGCCCTGC ACAAGCTGTG 2520
 TGTCCCTTGC CAAGTTACAT GACTTCACTA AGCTTCCCTG CCCTTATTTG TAAAATGGAA 2580
 ATACTAGCAC TGTTCCATAA GCTGGCCCCG TTGCACATAG TAATCGTAAT GGCAAATGCA 2640
 TGGAGCCAGG CGTTGTTTAA ATATTTTCCA ATGTTAACCC CATGAGCCAA TTACTGTTAT 2700
 CCCATTTTCC AGTGGAGGAA ATTGAGGCCC AGTGATGGTA AGTTACTTGC CTAGAATTAT 2760
 ATAGCTAGTA AGTGGCAGAG CCTAGATCCA AGCNGAGATG AKGTGGCTTT TTTKTTKTTK 2820
 TTKTCSAGAY AGAGTYTYRC TCTGTNGCCN ANGCNNGNGT GCNNTGGCNY NANCTNGGCT 2880
 YACNGCAACC TCCACCTSCT GGGTTCAAGC GATNCTCCAG CCTCAGMCTC CCAAGTAGCT 2940
 GGGATTACAG ACAYNTGCCA CCATGCCTGG CTAATTTTGT ATTTTTAGTA GAGACGGGTT 3000
 TCACCATGTT GGTCAGGCTG GTCTCGAACT CCTGACCTCA GACTATCCAC CCACCTTGGC 3060
 CTCCCAAAGT ACTGGGATTA CAGGCCACCA TGCCTGGTTT TTTGTTTTTT GTTTTTTTTT 3120
 TTTGAGACTG AGTCTTGCTC TGTCGCCTAG GCTGGAGTGC AGTGGCATGA TCTCGGCTCA 3180
 CCGCAACCTC TGCCTCTGGG GTTCAAGCGA TTCTCCTGCC TTAGCCTCTC TAGTAGCTGG 3240
 GATTACAGGC ACGCACCACC ATGCCCGGCT AATTTTTGTA TTGTTAGTAG AGACAGGGTT 3300
 TTTCCTGTTG GCCAGGCTGG TCTCGAAGTC CTGACTTCAA GTTATCTGCC CACCTCGGCC 3360
 TCCCAAAGTG CTGGGATTAC AAGCATGAGC CACCACACCT GGCNTTGATG TGGCCTTAAA 3420
 ATCCATTCTG TTGGCTGTGA CACTGTACTA TCTTACTATA TAGTAGGCAC TCAAAAAATA 3480
 TTTTTTATTG TTTTTGTGAG GATTTAATGA GACAAAACAT CTAATGTTTT TGGCACTTTG 3540
 AAAGCATTCA ATAAATGGTC ATCTCTTCCT TTCTCCCCCA GCATTGCTTT TAATGTGCAT 3600
 ATAAACATTG AACTATTGGC TATTACTGCA TCCAGAAGAA TGACAGACTA TCACCATGAG 3660
 CTGATGTCTA AAGATATTTC CAGATGGGTA GACCTGGAAA GGTATATTCA GGAACACTGG 3720
 GAGGAAGTAC TTAGTAGGTA TATTACTCAG TGTTTTAGGG GAATGTGCCC CCAGGTACAG 3780
 TCCTGCTTAA AAGGGCTGGA GGAGGTGGAT TGCTGGAGAA CTCTCATGTC CGCCATCAGC 3840
 ACACCACAGA GAGACCCCAC AGGTGTGGAG AAGGGGCAGG GCCTTCTTTT TCACAGCCAG 3900
 GAAAGCCAAA ATAAAGACTT GAGCAGGTGA ACTAATCACA ACTCAAAATG CCTTAGCTAT 3960
 CCCAGTAGGT GTGGAACCCA GCATCTAAGT AGTGTGTTCT GGCCAGAGAG CACCCCNACT 4020
 AGCATTGTCT GTGGTTCTTG CTTTCGTGGG TTGGAGATGA AGCTGTCATC ATAAACTCAG 4080
 AGGAAACCTT AAAATGTCTA GGGAGGGAAA GGAGTCATTC TAGTCTCTAG GCCAGAGCAG 4140
 AGCTGGAATC CTGCCCCATT CCTGCTTTTT CCACATCAGA AAAACGGTCA TGGTCAACCC 4200
 TGGKTGTCTT TGTGAGGNTG CAGGANCACT GCCGTCAAAG TGATTCCTCT GTGTGTCCCC 4260
 CCAAATCAAA TCCACGTTAA AATTAGAAAT CGATGGGGTT AAGATGGGAG AGTCAATGCT 4320
 AAACTCAGAG CAGCACAAGT GAAGCCTTCT CTTTTGACAG CCTAGGCTAG GTCCACAGCC 4380
 TCTCTTGCAA AGATTAAAGT AAAAGGCTAA CTGACTGTGG TCTCAACTTT ATCATATTTG 4440
 CCAATTAGGA ATTCTTCCTG TGGTAGATAT CTCTTAGGAG TGTATTGTTG TGAACAGAGA 4500
 CACAGTATTT GGATACATYT ATTWTWKAWA TWTTCTCCAG YKACAYATTK CAMCACATNT 4560
 ACCYYCRGGC RMATCTCAGA RANAWACAMA CTCTTWCTNM TCMATGRAGT AACATGGAKW 4620
 TANAGGGRNG TGTTCCYGGR RGTGTGACTC TGWRCTGTGT ATATCAAGAR KCWCTGGGAT 4680
 GCTTATAAAA TGARAATATC TGAGMCCMMC CCCAGACCYY CTGWATAACG AYATGGGGGT 4740
 GAGTGAGACT GGGGAGTCTC CATTTTAAAC ATGTACACAG TCATTCTCTA NTTTGTTTTT 4800
 TTTTTGCTGC TGTTGNTGAG ACAAAGTCTC ACTCTGCTGC CTAAGCTGGG GTG 4853
 (2) INFORMATION FOR SEQ ID NO: 23:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 5658 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: IPF1 gene; contig 2.
 (ix) FEATURE:
 (A) NAME/KEY: transcriptional start
 (B) LOCATION: nucleotide 2002
 (ix) FEATURE:
 (A) NAME/KEY: translational start codon
 (B) LOCATION: nucleotides 2106 through 2108
 (ix) FEATURE:
 (A) NAME/KEY: first coding region
 (B) LOCATION: nucleotides 2106 through 2511
 (ix) FEATURE:
 (A) NAME/KEY: intron 1
 (B) LOCATION: nucleotides 2512 through 5858
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23
 GCGGCCGCAG ACAATGGACT CATTGACGCG GGCGCACAGA GATTAGGTGC CCGGGCCCCA 60
 CGGCCTTTCA CTCTCTGCCC CCACCAAAAC TGCAGGTGGC ACTTTTCCCT GAAGAAATCA 120
 CCGAAACTCT CCCCGCCCGC CGCCGTGCTT CCCTTGGCCT GTCTTCGGTG ACAGCAAGGG 180
 CATCTACAAG CCAGGGAGCC ACACATCAGA GCCACACACA TGAGGCCACA GGCCAGGAAG 240
 CCACACACAT GAGTTCTCAC ACATCGAAGC AAATGCTGAG ACTATACCGG CCTCCCGCAA 300
 GAGAAGGAAT CTGCAGAGAA GTCCACATTC CCGACTGGCT TTTTTGTCTT TTAATAAAAA 360
 TAAAGAGTCC TCAGAATAGA GCACTCAATC CGCACTTAAA ACACAAATAT TTCCAGTAGG 420
 AAGAAAGAAA TACATTAAGA TTGACTCTCA AAGGTCTGGA TGTTGGTGCG TTGTTTAGGA 480
 AAGGCGGAGC AGTGATTTTT CTCTGGCTTG GTTCTTTGTG GTTCAAGCTC TCTCTTGCTG 540
 AGACGTTTCT GCAAAGCTGT CTAGTTTTTC TAAAACCCAC GGTGGCGTTT CGAGAAACGT 600
 CCTCATTTCG GGAGTATCCA GTCAGAGGCT GGTCAGGCCG CCAGGCCTAG CGGATCATGC 660
 CCAGGCAGGG GGTTGGACGT CACCGCCACC CGGAGGTCAT CCATCCGCGT CAGTGGGTGC 720
 AGAAAAAGTG GCCCTGTTTA AGTCAGGACC CCAGAATGCC CAGAAGTTAC TGAATGGTTT 780
 GAAGCCAAGC ACAGATGTTA TCATGGAAAA TGCAGCGTTT TTATTTCTTT TTCTAAATAT 840
 GTAACTCTTC CTCCACTTCC CCCTCTCCTG CTTGCCTTAT TTCAATTGCA AGCAGAAGAG 900
 AGTGAGTGTT CTCTGCCGGC AAACTCCGCC AGGGTCCCGG CCCGTAGAGA GTCGTCAAGG 960
 GTCTGGAACC CCCGTGCCAA CACCTGCCCC TGCTTCGCAG CCCCAAGAGG AAGGCCGCGT 1020
 CTTTCCCCCT CGCTGTATTG GGAAGCTACG TTCCGGGCTG GCCAAATGGG CCCCAATTTT 1080
 CCAAAACCCA AATTTGTAAT ACCCTTCAAT TTTTTAAAAA AAAGAATTTA AAAAAGTCTC 1140
 TGTGAATGCT TCAGAAGTTA CCGTTTACAC CCCAGAAGTA CTTGCAGCAC ATCCACAAGT 1200
 AAAAACACAC AACGAATGCC AGAGTTTCGT GTGTTTTTTA ACCGACATCT TTGTGGCTGT 1260
 GAACAAACTT CATAAATAAA ATAGAATCAA ATGCTTCTGA CCTAGAGAGC TGGGTCTGCA 1320
 AACTTTTTTT TTATCGTATT CCGCAACAGT TAAATAAAAA ATTAAAAACT CAACATGTCT 1380
 CCTTGTAAAC TACATCAATT AACAAACACA CTATGTCCAT TATCAAATAT AATAGAAAAA 1440
 ATATAGGAAA ATAGAAAATA GAAAAATATA GGAAAATAGA AACTTTTAAG CCACGGTGAA 1500
 AATGTTTCTA TAAATGAGTG GTTCTAATGT TTTCGTGAGC GCCCATTTTG GGGAGCACCG 1560
 CCAGCTGCCC GTTCAGGAGT GTGCAGCAAA CTCAGCTGAG AGAGAAAATT GGAACAAAAG 1620
 CAGATGCTCG CGGGTACCTG GGCCTAGCCT CTTAGTGCGG CCAGCCAGGC CAATCAACGG 1680
 CCCCCGCTGA ACCACGTTTG GGCCCGCCTN GGGAGNCTTG GTNNNNNNNN NNNNNNNNNN 1740
 NNCCGNGNTG GCTGTGGGTT CCCTCTGAGA TCAGTGNGGA GCTGTCAAAG CGAGCAGGGG 1800
 TGGCGCCGGG AGTGGGAACG CCACACAGTG CCAAATCCCC GGCTCCAGCT CCCGACTCCC 1860
 GGCTCCCGGC TCCCGGCTCC CGGTGCCCAA TCCCGGGCCG CAGCCATGAA CGGCGAGGAG 1920
 CAGTACTACG CGGCCACGCA GCTTTACAAG GACCCATGCG CGTTCCAGCG AGGCCCGGCG 1980
 CCGGAGTTCA GCGCCAGCCC CCCTGCGTGC CTGTACATGG GCCGCCAGCC CCCGCCGCCG 2040
 CCGCCGCACC CGTTCCCTGG CGCCCTGGGC GCGCTGGAGC AGGGCAGCCC CCCGGACATC 2100
 TCCCCGTACG AGGTGCCCCC CCTCGCCGAC GACCCCGCGG TGGCGCACCT TCACCACCAC 2160
 CTCCCGGCTC AGCTCGCGCT CCCCCACCCG CCCGCCGGGC CCTTCCCGGA GGGAGCCGAG 2220
 CCGGGCGTCC TGGAGGAGCC CAACCGCGTC CAGCTGCCTT TCCCATGGAT GAAGTCTACC 2280
 AAAGCTCACG CGTGGAAAGG CCAGTGGGCA GGTAAGCCTG GCTCCCCACC CCTTTCTCCT 2340
 TTCCGGTTCT CACCCGGNNG CCTTACCTCC AAGCGcTCCC AGGAGCCTTC TCTCTGTTCC 2400
 CGGCGCCTTG GATTATCCCG GGTCGGACTA AACTACATCA GGGAGCTACC GAGCCCATCC 2460
 CTCACAGCAG TGCTTCTCTA GTCCAGTTTG AAGCATCTTT CCCACCCAGC TCTCCTGGGA 2520
 GTGTACACTC CTTCCTTCCC TGTTCGCTGA GCCCATCTTC GCCCCAGGAG CCCGCGCTCC 2580
 CAGCGCCATC CTTAGAGAGC CGAGGCTGAG TCCTGCTCAG GGCTTCGGAC ACTACAGATC 2640
 CTCCTCCAGC AGGGGATCCG GGAACCCAGG ACTCCTTGGT AGTGCACATC GAGGAAGCCG 2700
 AGTAAGGACA TGGGTGCCTC GGACCCAGGC CCCAGATCGC CTTCGGAGCC CCGGAGCCCC 2760
 TCACTTCCCG CGCTTCGTTA AGGAAGGGCA GGCATCTARG GGCGCCASGT AGGTGCAGAA 2820
 AGGCAGGGAG GGAAAGGAAA CTGCACCCAA CCCAGCAGTG TCCGGCTGCC CTGGTTGTGG 2880
 AAACAGGATA GATAAAGAGG AAGGGGCTGG GGCAAGGCGG GGGCTCACCG CGAGGCTGAA 2940
 AGCCGGCCTC TCAACNTCAG AGCCTGGCAG CTAGGAGAGC AATCTGAGAA GCGAATTCGT 3000
 TTTTCACCAA CCGAAAGCAA TTGAAGCTGT CTCCCCGCAC CGCTTCCCAG GAAGTAATTT 3060
 TTCAGGAGAT GGGCGCTCCC TGCCTAACTG GTGGGGAAGC GAAAAGCCTG NTTCCTGCGG 3120
 CCCTCCGCGC CGGCANAAAA CAGAANGTCT TTCCCGGAAC CGGGARCCGG ARGCACGGGG 3180
 TANCCCCCGG GTCCTTTGCG GCCCCNCGCG AGCGGCAANT TCCGGCGCGG CCTGTGTCGT 3240
 CGCCGCTACT CACTGTCATC GCTGCCGTGC CTCAGCCACT TCTGGTCACA CCTGCACCGC 3300
 AAATAGTTGC CTTTTCCTTT CAACTGGCAG CCGGGAGTAG GGGGAAGCAG CTCGAGCCGG 3360
 CGTCCCCCGG CCCACCCCGA AAATCCTCAG CGCCCATCTG CGGGGTCTGG CCAGCCCTGC 3420
 CTGACACTGA CCCCAGGCGC AGCCAGNAGG GGCTTTGTGC GGGANANGGA AGGGGAACCC 3480
 ATCTTTNNTN NGGTCCACGG GACTCTCTTC TTCCTAGTTC ACTTTCTTGC TAAGGCGAAA 3540
 GTCCTGAAGC AGGACAAAGG CTGAACTGCG CTGCAATCGT CCCACCTCCA NCGAAACCAA 3600
 GTTGACAGGG GCGCCCCAAG CTGCCACGGC GCCTCTGCAA ATTTATCCAG CTCGCGCAGC 3660
 CCGGGCCAAA GGCCTTGAAG TCTCCGGAAA TGCGGGGTTC TTAGGAGGCG GGAGGACAGT 3720
 CCCTCGAACA AAGGTGGGGG GCTCCTCGTC CTCACCCAGT TTTCTTCCAG GGCTGCCTCC 3780
 CCTCCAAACC TCTCTTCTGG CCTCCTAGGC CCTSGGAGCT CCTGCTTTCC CACCYTGGGC 3840
 CTTCYTCAGG AAATGGGMGA CATCAGGGTC CCGAAAGAGG ATTTGTGAGG TGGAGTAACT 3900
 TCCYTATCCC AACCCAAGGG GTGATACMTM TGMTMTGKAK GAYTTKGGCT TAGGCTGACC 3960
 CAAGAAGCCA GAAAGTAAAA CCAGAAGGCA AATCAGCAGC CTTGGCGAGG GTTCGGGGAC 4020
 CCAAGGAGGG CGACACTCTC GGGCTGGAGT TGGCCCCAGG CCTTTGCTGG CGCCCTCTAA 4080
 CCCGCTGCAT GCTCGACTTT CGGGGAAGGA GACGACCTCC CCTCTCTTCC CCTGGAAGCC 4140
 GTCTGCGGGG CCGGCTGCTA TCCCCGCGTT CCTTTAGGGG AAACTTCGAT GGAGCCGAAA 4200
 TTCAAAAATT GCAAACCCAC CTGCCCCTGG GAAGAGCGAA GTGACAAAAG GGCTCTCMAY 4260
 GGNCAGTACG AATTTGAATG TTAATGACAA CAGAGGTTTT GAAAAACATT GACCCCCAAA 4320
 TGCTTCAGCA GCGCTGTCCA GNTGGCACNT AAANTGCATC ANTNTGCGCC TTGGGGAAGG 4380
 GCCCAGGCTT GGNGACATNG ACATTTTCCC ACCATCCTCA ACCTCCACCC CTGCCGCGTC 4440
 GCGCTGAGCA CAGGTCCCCC GGGAATAGTG CACCCCAGGA AGTCTCTCCC TGAGCAGTCT 4500
 CTCGCAGGGA CTTCACGAAG CCCTCTCGCA GGGACTATAC GAAGCCCGCA GCCTAAGGCA 4560
 GGAACCCAGA GACATGTCGG TTTAATGTAA AAACTTTGGA GAGCCTTTCA AAATGTTTAT 4620
 TGAAGGCCCG TCTCGCTTCT CTCCCAGGCG TGGGATGCCA GGTAGATTCG GGGATGCCCC 4680
 CAGGGAGTAG AACTCTCCCT GGACTAGGGT TTGAGCCTCT GCTTCAGCTT NTGGCGCCTC 4740
 TTCTCGACCT TGGGGGGAAA CCCAGTCAGG TTCTCTCGGG AAATTAACCC CGCCCCCAAA 4800
 ACACACACAT TCGCCTTTCA ATTCGTTAAG GCTNAGCCAA CATTCACAGG AGAAATGTCC 4860
 CCTGCCTTTG CTGTAAGACA AGCCTCTCCC CGGAACTTTG GTGGAACTTC CCGCGCCAGC 4920
 GTCCACAGCC TGGGTGCAGT CAGTATTTTC CACAGAAAAG AAAAGATTGG GACTTGGCTG 4980
 AGCGCAGCGG CAAACAGTGA ATGTGGGTCT CCAACTTCNT GGGNCAGGGG TCCNGTTGCC 5040
 TCTTGGAGAC ANGAGAGGCT TGTTTGTGCA CCATACCACC TCNTCCGTAG GGCTGTNGGT 5100
 TNTGCAGGTG GGTAAGGGCC CANGTGTCTT CCCCTCAACA CCTCTGAGGG CATTTGGGAT 5160
 CCCAGRGCGT AGATTCNGGA GCTGCCAGAG TTNTGCCTTG GCCAACGGAC CCCCAGAACA 5220
 ATATTCTTTC ANTTTNGCGG GCAGAAGTCC GGCTGAAGTT AAAACAATTA TGGAGAATTT 5280
 GCTGGTCTCA GGTTGGGACT AATTACGATA TAACTATAGA GAGAGGAAAC ACATGGTCAG 5340
 ATATAACAAA ATGTGTCACA GTCTCCATTA GCACAAAGAT TTTCAAACTG CAGGTTGCAC 5400
 CCATTCGCAG GTCATAAAAT CAATTTACTA GGTTGAGATT AGTATTTTTT AAACGAAATA 5460
 GCAGATAATG GAGAGAAAAG TAGATAGCAT CATACGTGGT AAACGTTTGT TTTATGTCCT 5520
 TAAGATTTGT CAGTATAACT GACCTGCAGT GTCCGTGTGT GAACTACACA ACGATCCGAA 5580
 ATGTATTTCT CACATTTGTG GGTCACCATC AGGAGGTTTT TTTTAGCCCT GGATTAAAGG 5640
 CGTTGNCTTG CCTTTGTA 5658
 (2) INFORMATION FOR SEQ ID NO: 24:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 874 nucleotides
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: genomic DNA
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: IPF-1 gene, contig 3.
 (ix) FEATURE:
 (A) NAME/KEY: intron sequences
 (B) LOCATION: nucleotides 1 through 359
 (ix) FEATURE:
 (A) NAME/KEY: second exon
 (B) LOCATION: nucleotides 360 through 874
 (ix) FEATURE:
 (A) NAME/KEY: second coding region
 (B) LOCATION: nucleotides 360 through 802
 (ix) FEATURE:
 (A) NAME/KEY: stop codon
 (B) LOCATION: nucleotides 803 through 805
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24
 GGATCCAGCC GGTTTAACTA TTATATACAT TTAAATCAAC ATCAATCAGC TGATTAACAC 60
 CGATTATATG AACGCATTCA AGGACCACTC ATTGGCAGAG CCAAGCTTAG GCTCACGGCG 120
 AGAGCTGACT CGAGTTTGGT CTCCAATAAA AAGGCTATCT TTATTAGGAA GGGCTTGAGT 180
 TACTAGGGAA GAGCTTCGCG CGCCTACACT AGGCGCTGAA ATGGGATGCT GGGGCTTGGT 240
 GGCTCCGCGG AGCAGCTGGT AGGCTAGGCT CCCTGGGCCC CCCTTGAAGG GTTTGGGCTG 300
 CGTGGGTGGG GGCTGTGCGG GGCTCCGGGG GCCACACTCA CGCCCTGTGT CGCCCGCAGG 360
 CGGCGCCTAC GCTGCGGAGC CGGAGGAGAA CAAGCGGACG CGCACGGCCT ACACGCGCGC 420
 ACAGCTGCTA GAGCTGGAGA AGGAGTTCCT ATTCAACAAG TACATCTCAC GGCCGCGCCG 480
 GGTGGAGCTG GCTGTCATGT TGAACTTGAC CGAGAGACAC ATCAAGATCT GGTTCCAAAA 540
 CCGCCGCATG AAGTGGAAAA AGGAGGAGGA CAAGAAGCGC GGCGGCGGGA CAGCTGTCGG 600
 GGGTGGCGGG GTCGCGGAGC CTGAGCAGGA CTGCGCCGTG ACCTCCGGCG AGGAGCTTCT 660
 GGCGCTGCCG CCGCCGCCGC CCCCCGGAGG TGCTGTGCCG CCCGCTGCCC CCGTTGCCGC 720
 CCGAGAGGGC CGCCTGCCGC CTGGCCTTAG CGCGTCGCCA CAGCCCTCCA GCGTCGCGCC 780
 TCGGCGGCCG CAGGAACCTC GATGAGAGGC AGGAGCTGCT CCTGGCTGAG GGGCTTCAAC 840
 CACTCGCCGA GGAGGAGCAG AGGGCCTAGG AGGA 874