EP0959907A1 - The butyrophilin gene promoter and uses thereof - Google Patents

Abstract

The DNA sequence of the mouse butyrophilin gene and its promoter is disclosed and analyzed. In addition, expression of the mouse butyrophilin gene is characterized. Further, use of the butyrophilin promoter for expressing polypeptides in the milk of a transgenic animal and for screening substances for carcinogenicity is disclosed.

Description

THE BUTYROPHILIN GENE PROMOTER AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates generally to the butyrophilin gene promoter. More

specifically the present invention relates to the use of the butyrophilin gene promoter for the

production of heterologous proteins in the milk of transgenic animals and for the detection of

carcinogenic substances. Applicants hereby incorporate by reference the subject matter of U.S.

Serial No. 60/022,563.

BACKGROUND OF THE INVENTION

Butyrophilin is the major integral protein associated with the fat-globule membrane

(FGM) in the milk of many species and is believed to play a role in the mechanism of milk

secretion. See Franke et al. , J. Cell Biol. 89: 485-494 (1981); Jack and Mather, J. Biol.

Chem. 265: 14481-14486 (1990); and Jack and Mather, /. Dairy Sci. 76: 3832-3850 (1993);

each of which is herein incorporated by reference. Expressed on the apical surfaces of

mammary epithelial cells, butyrophilin is a type I glycoprotein, comprising a glycosylated

exoplasmic domain, a membrane anchor approximately in the middle of the sequence, and a

long cytoplasmic tail.

Butyrophilin is a member of the immunoglobulin superfamily (IgSF) (Gardinier et al.,

J. Neurosci. Res. 33, 177-187 (1992)), with closest structural homology in the exoplasmic

domain to the B7.1 (CD 80) and B7.2 (CD 86) receptors (Linsley et al., Protein Sci. 3: 1341- 1343 (1994)). Hallmarks of these proteins are two exoplasmic immunoglobulin-like domains; one of the variable (V) or intermediate (I) type (Williams and Barclay, Ann. Rev. Immunol. 6,

381-405 (1988); Harpaz and Chothia, J. Mol. Biol. 238, 528-539 (1994)) close to the N-

terminus; and one of the constant (C) type (Williams and Barclay, 1988) close to the membrane

anchor. Other proteins that are homologous with butyrophilin in the exoplasmic domain

include myelin oligodendrocyte glycoprotein (MOG), a component of the myelin sheath

(Gardinier et al., 1992), and the chicken B-G antigens associated with the avian major

histocompatibility complex (Miller et al., Proc. Natl. Acad. Sci. U.S.A. 88: 4377-4381

(1991)). MOG and the B-G antigens have shorter exoplasmic domains with one V-set

immunoglobulin-like fold (Gardinier et al. , 1992; Miller et al , 1991). The inclusion of

butyrophilin in the IgSF and the B-G antigen system suggests that butyrophilin has immune functions.

The C-terminal cytoplasmic domain of butyrophilin is similar to the C-termini of a

group of proteins that contain zinc finger and coiled-coil domains. These proteins may bind

nucleic acids or proteins (Bellini et al. , J. Cell Biol. 131 : 563-570 (1995)) and include ret

finger protein (RFP) (Takahashi et al., Mol. Cell Biol. 8: 1853-1856 (1988)), nuclear antigen

A of Sjόgren's syndrome (SSA/Ro) (Chan et al, J. Clin. Invest. 87: 68-76 (1991)), Xenopus

nuclear factor 7 (XNF7) (Reddy et al, Develop. Biol. 148: 107-116 (1991)), PwA33 from

Pleurodeles waltl (Bellini et al, EMBO J. 12: 107-114 (1993)), and acid fmger protein (AFP)

(Chu et al , Genomics 29:229-239 (1995)). At the DNA level, this homologous region

encompasses an exon, named B30.2, which was mapped together with the MOG, RFP and

butyrophilin genes to the human MHC class I region of chromosome 6 (Vernet et a , J. Mol. Evol 37: 600-612 (1993)). Based on these observations, Vernet et al (1993) suggested that

the butyrophilin gene evolved in the MHC by the shuffling of exons between an ancestral MOG gene which gave rise to the exon encoding the I-set immunoglobulin-like domain of

butyrophilin, and an ancestral RFP gene, which gave rise to the B30.2 region of the

butyrophilin gene.

Butyrophilin is specifically expressed in mammary tissue, with expression being

maximal during lactation. This mammary-specific expression of the butyrophilin gene is

assumed to be under the control of the butyrophilin promoter. Since butyrophilin constitutes

a significant portion of the total protein associated with the milk FGM of many species, i.e. ,

more than 40% of the total FGM-associated protein in bovine milk is butyrophilin, the

butyrophilin promoter is an attractive mammary-specific promoter for producing heterologous

protein in the milk of transgenic mammals.

Promoters of other mammary-specific genes, i.e. the casein, whey acidic protein, α-

lactalbumin, and β-lactoglobulin genes, have been used to direct the production of foreign

proteins in the milk of transgenic animals. Recent analysis of these mammary-specific gene

promoters has led to the identification of a number of potentially important regulatory elements

which mediate the lactogenic response. These elements include binding sites for the following:

CTF/NF1 in the β-lactoglobulin (Watson et al , Nucl. Acids Res. 19: 6603-6610 (1991)) and

whey acidic protein genes (Li and Rosen, Mol. Cell Biol. 15: 2063-2070 (1995)); Oct 1 in the

bovine α_s2-casein gene (Groenen et al, Nuc. Acids Res. 20: 4311-4318 (1992)); a single-

stranded nucleic acid binding protein which negatively regulates the β-casein gene (Altiok and

Groner, Mol. Cell Biol 14: 6004-6012 (1994)); Ets-related proteins which stimulate (Welte

et al , Eur. J. Biochem. 223: 997-1006 (1994)), and unidentified factor(s) which negatively

regulate, the whey acidic protein gene (Kolb et al , J. Cellul. Biochem. 56: 245-261 (1994)),

and a pregnancy-specific protein which modulates progesterone-mediated repression of the mouse β-casein gene (Lee and Oka, J. Biol Chem. 267: 5797-5801 (1992)). Several genes,

including the most intensively studied rodent β-casein gene promoters, contain C/EBP

(Doppler et al , J. Biol. Chem. 270: 17962-17969 (1995); Raught et al. , Molec. Endocrinol

9: 1223-1232 (1995)), YYI (Meier and Groner, Mol Cell Biol 14: 128-137 (1994); Raught

et al , Mol. Cell Biol. 14: 1752-1763 (1994)), MGF/STAT5 (Watson et al. , Nucl. Acids Res.

19: 6603-6610 (1991); Groenen et al, 1992; Wakao et al , EMBO J. 13: 2182-2191 (1994))

and glucocorticoid response elements (Raught et al, 1995). In addition, the promoter of the

housekeeping gene, βl,4-galactosyltransferase, contains binding sites for AP-2 and CTF/NF1,

which regulate the synthesis of a mammary-specific 3.9 kb transcript (Rajput et al. , J. Biol Chem. 271 :5131-5142 (1996)).

The basis for mammary-specific expression is poorly understood in any system.

A so-called "milk-box" sequence, first identified in the proximal α-lactalbumin gene promoter in several species, is also conserved in many of the casein genes (Laird et al , Biochem. J. 254:

85-94 (1988)), and encompasses binding sites for YYI, STAT5 (Meier and Groner, 1994;

Raught et al. , 1994) and C/EBP isoforms (Doppler et al. , 1995; Raught et al, 1995). Also

there are three conserved sequences in the casein genes referred to as blocks A, B, and C

(Yoshimura, M. and Oka, T. , Gene 78, 267-275). Raught et al. (1995) have recently suggested that casein gene expression is regulated by composite response elements (CoREs)

comprising STAT5 and glucocorticoid response elements and C/EBP binding sites.

For the first time, the inventors have cloned and sequenced the mouse butyrophilin

gene, including its promoter region and have found that the promoter sequence has no

significant similarities with the published sequences of these other mammary-specific

promoters. Analysis of the butyrophilin promoter sequence showed that the butyrophilin promoter

contains many potential regulatory elements associated with immune system genes including

cx- and γ-interferon response elements, and consensus sequences for TCF-1 and PU. l . (PU. l

is a macrophage and B cell-specific transcription factor related to the ets oncogene. See

Klemsz, et al. , Cell 61 : 113-125 (1990)). In addition, the inventors have found that the

proximal region of the butyrophilin promoter contains a repeat element of three granulocyte-

macrophage colony-stimulating factor (GMCSF) sites which in the same context has been

shown to regulate the mitogen-inducible expression of GMCSF in T cells (Nimer et al. , Mol.

Cell. Biol. 10: 6084-6088 (1990), herein incorporated by reference). Thus, the butyrophilin

promoter is also useful for the detection of carcinogenic substances.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides the sequence of the 5' flanking region and

transcriptional unit of the mouse butyrophilin gene (Btn). In particular, it provides the Btn

promoter and transcriptional regulatory elements contained therein.

Accordingly, an object of the invention is an isolated and purified DNA fragment

comprising a DNA sequence encoding a polypeptide having the biological activity of a

butyrophilin protein.

Another object of the invention is an isolated and purified DNA fragment comprising

a DNA sequence having the biological activity of a butyrophilin promoter.

An additional object of the present invention is a rDNA construct for expressing a

polypeptide in the mammary gland of a mammal. The rDNA construct comprises a butyrophilin promoter operatively linked to the DNA sequence encoding a desired polypeptide. The rDNA construct may also have a DNA sequence encoding a signal sequence operatively linked to the DNA sequence encoding the polypeptide. Preferably, the signal sequence is a

milk protein signal sequence. The DNA construct may also include the transcriptional unit

and/or 3' flanking sequence of the butyrophilin gene.

It is a further object of this invention to provide a transgenic animal which produces a

desired polypeptide in its mammary gland. This is achieved by introducing a rDNA construct

comprising a butyrophilin promoter operatively linked to the DNA sequence encoding the

polypeptide into at least the mammary epithelial cells of the mammal. Alternatively, the rDNA

construct may be introduced into a germ line of a mammal, thus subsequent generations will also express the desired polypeptide in their milk.

Another aspect of the present invention is the use of the mitogen-inducible elements in

the butyrophilin promoter to detect mitogenic properties of potential carcinogens from a variety of sources. For example, substances found in the environment or isolated from food sources

could be tested for carcinogenicity. The mitogenic properties of a substance are assessed by

detecting activation of the butyrophilin promoter in cells exposed to the substance, either by

detection of butyrophilin mRNA or protein, or by detecting expression of a reporter gene under

the control of a butyrophilin promoter.

Yet another object of the invention is diagnosis of disease states such as breast cancer

by screening mammary and nonmammary tissues of nonlactating animals for the expression

of butyrophilin. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the λBtnl clone, showing the location of subclones prepared from λBtnl which were used to generate the sequence of the mouse butyrophilin gene and 5' flanking region.

FIG. IB is a schematic representation of the structure of the mouse Btn gene, showing the location of the exons and introns.

FIG. 1C is a schematic representation of mouse butyrophilin cDNA, showing the location of the cDNA subclones used to sequence mouse butyrophilin cDNA.

FIG. 2 A-C shows the location of putative regulatory elements in the proximal 5' flanking region of the mouse butyrophilin gene.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides the sequence of the mouse butyrophilin gene, and approximately 4.6 kb sequence of its 5' flanking region, which is also referred to as the butyrophilin promoter. These sequences were obtained from a clone isolated from a murine genomic library

as described below.

Example 1 : Cloning the Mouse Butyrophilin Gene

Screening of genomic library and cloning of λBtnl. A 129 ES cell genomic library in Lambda DASH^* (Stratagene, La Jolla, CA) was screened with a 2.3 kb Xhol-Xb fragment of cDNA encoding bovine butyrophilin (Jack and Mather, 1990). Plaque DNA (total of 500,000 pfu) was tr.ansferred to nylon membranes (Dupont, Boston, MA), denatured in 0.5N NaOH, neutralized and cross-linked to the membranes by exposure to ultraviolet light using a UV Stratalinker^® 1800 (Stratagene). Membranes were incubated for 2 h at 42O in prewash solution (Sambrook et al. , Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring

Harbor Laboratory Press, NY, (1989)), followed by 6 h at 42^° C in pre-hybridization solution,

and were then incubated overnight at 42°C in hybridization solution with the bovine

butyrophilin cDNA fragment which had been labelled with [α-³²P]-dCTP to a specific activity

of 10⁹ cpm/μg by the random priming method (Feinberg and Vogelstein, Anal. Biochem. 132:

6-13 (1983)). Filters were briefly rinsed with 2X SSC (Sambrook et al, 1989) and then

washed three times at 55° C with 2X SSC containing 0.1 % (w/v) SDS, for 20 min each time, and the cDNA bound to the membranes detected by exposure to X-ray film, overnight, at -80°

C. One potentially positive plaque was detected from a total of 500,000 pfu's screened and

this cloned DNA was designated λBtnl , which has been deposited with the American Type Culture Collection as ATCC designation 97513.

To confirm that λBtnl contained the mouse butyrophilin gene, samples of the cloned

DNA (λBtnl), mouse, and bovine genomic DNA were digested with either EcoRl or Hindlll

and subjected to Southern analysis using the [³²P] -labelled 2.3kb Xbal fragment of heterologous

bovine cDNA as a probe. In each case, digestion with the restriction endonucleases generated

a characteristic pattern of DNA fragments which hybridized to the [³²P]-labelled probe.

Similar patterns of radiolabelled bands were detected in the genomic DNA and λBtnl samples

(data not shown). As the sequence of λBtnl became available, a mouse cDNA probe, mcDNA3, encoding the 3' end of exon 3 through the first 396 bp of exon 7 (see FIG. IC) was

prepared by RT-PCR. A Southern blot, similar to those described above and probed with this

homologous cDNA, confirmed the identity of λBtnl (data not shown).

Example 2: Sequencing the Mouse Butyrophilin Gene Subclones spanning over 14 kb of λBtnl DNA were prepared (see FIG. 1A) and

sequenced on both strands using the finol sequencing kit from Promega Corp (Madison, Wl).

Autoradiographs were scanned with a Molecular Dynamics Computing Densitometer and the

sequences read using the Image Quant^*, Version 3.30 software package (Molecular Dynamics,

Sunnyvale, CA). The computer program MACAW (Schuler et al, 1991) was used to compile the full-length sequence from the sequencing gels and the sequence is shown in SEQ ID NO: l.

The entire Btn sequence has been deposited in the GenBank Data Base under Accession No.

U67065. The butyrophilin promoter is contained within the first 4,693 nucleotides of SEQ ID

NO: l . The proximal part (first 1750 nucleotides) of this region is shown in SEQ ID NO: 2

and schematically illustrated in FIG. 2, with the nucleotides being renumbered in conventional

format, i.e. , where the most proximal transcriptional start site (see below) is designated + 1.

Example 3: Expression of the Mouse Butyrophilin Gene

Mapping the 5 'end of mouse butyrophilin mRNA. The transcriptional start sites were

identified by primer extension analysis using a ³²P-labelled primer having the sequence, 5'-

GGGCTCTGTATTTCCCCTAC-3' (SEQ ID NO:3), and total RNA from day 14 lactating

mammary gland. This primer extension assay was adapted from Roussel et al. (DNA Celt Biol

14: 777-788 (1995)), which is herein incorporated by reference. Three major labelled products

were obtained from this primer extension experiment, suggesting that transcription of Btn is

initiated from at least three sites, at nucleotides -83, -19 and + 1 (FIG. 2) (residues 4611 , 4675, 4694 of SEQ ID NO: l) with the most frequently used site at nucleotide T, designated

position -83 in FIG. 2.

All three transcription start sites are close to or within the context of, the initiator

element 5'-YYA_{+ 1}NWYY-3' (Javahery et al, Mol. Cell Biol. 14:116-127 (1994), herein incorporated by reference) which can mediate the initiation of transcription in genes lacking

conventional TATAA and CCAAT boxes. Two of these sites, at positions -83 and -19, contain

one and three mis-matches, respectively, from the consensus sequence, and the site at position

-83 is two nucleotides downstream of the more usual A_{+ 1} start site. The most proximal start

site at nucleotide + 1 is within a perfect consensus, although paradoxically it does not appear

to be the most frequently used.

Although Btn does not have conventional TATAA elements, two AT-rich regions, 5'- TGTAAAT-3' at position -49 (nucleotides 4645-4651 of SEQ ID NO: l), and 5'-TCTAAA-3'

at position -106 (nucleotides 4583-4588 of SEQ ID NO:l) are within 20-25 nucleotides of the

two weaker initiator elements. In common with other genes these regions may cooperatively

strengthen the initiation of transcription via the TATA- and initiator-binding proteins (Javahery

et al, 1994), and this may explain why the start site at position -83 appears to be the most

frequently used site. Interestingly this latter site is closest to the sequence 5'-TCTAAA-3'

(position -106 of FIG. 2), which is a characteristic TATA element in many human MHC class

I genes (Le Boutellier, Crit. Rev. Immunol. 14: 89-129 (1994), herein incorporated by

reference). In addition, it should be noted that many of the milk-protein gene promoters have

rather similar atypical TATAA boxes, including the sequence 5'-TTTAAAT-3' in the rat and

mouse whey-acidic protein genes (Campbell and Rosen, Nucl. Acids Res. 12: 8685-8697

(1984)) and many of the casein genes (Yu-Lee et al. , Nucl. Acids Res. 14: 1883-1902 (1986)).

Btn also lacks typical CCAAT elements in the expected context approximately 50

nucleotides upstream from TATA sequences (Breathnach and Chambon, Ann. Rev. Biochem.

50: 349-383 (1981), herein incorporated by reference). However, there are several potential

CCAAT-like elements (double underlined in FIG. 2), including the sequence 5'-ACAAAGT-3' (nucleotides 4597-4603 of SEQ ID NO:l), which is within 50 nucleotides of the proximal

TATA box, and the sequences 5'-CCATTT-3' and 5'-CATTT-3' (nucleotides 4546-4551 and

4533-4537 of SEQ ID NO:l , respectively) which are 30-40 nucleotides upstream of the distal

TATA box. Of the milk-protein gene promoters sequenced to date, none have conventional

CCAAT boxes.

Mapping the 3 -end of mouse butyrophilin mRNA by RT-PCR. The polyadenylation

signal sequence in Btn was identified by using the RT-PCR to amplify four regions of cDNA

around the first potential polyadenylation (poly A) signal sequence (nucleotides 13091 - 13096

of SEQ. ID NO. 1) after the stop codon in Btn. Amplified products of the expected size were

obtained with primers 5' of the putative poly(A) signal sequence, while no RT-PCR products

were obtained with the primer pairs surrounding this poly (A) signal sequence or encompassing a region 3' of nucleotide 13,199 (data not shown). These data suggest that the first potential

polyadenylation signal in Btn is the preferred termination signal and that the 3 ' end of the

transcripts lies between nucleotides 13,097 and 13,199.

The predicted 5'- and 3'- boundaries of Btn lead to estimates of approximately 8.40-

8.57 kb for the sizes of the initial gene transcripts, and values of 3.50-3.68 kb for the sizes of

the processed mRNAs. These latter estimates are in good agreement with a value of 3.7 kb

for the size of mouse butyrophilin mRNA determined by Northern analysis of total RNA from

lactating mouse mammary gland using mcDNA3 as an oligonucleotide probe (data not shown).

Sequence analysis of the butyrophilin gene sequence identified single inverted repeats

in the 5' untranslated region (5' -UTR), and 3 '-untranslated region (3' UTR). Interestingly,

the repeat sequence in the 5'-UTR (nucleotides 4807-4814 of SEQ ID NO:l) is the exact

complement of the 3 -UTR sequence (nucleotides 12,556-12,563 of SEQ ID NO: l), suggesting that these sequences play functional roles in the synthesis, stability or regulation of butyrophilin transcripts.

Translation of Mouse Butyrophilin mRNA. The predicted murine butyrophilin amino

acid sequence was derived after verification of exon/intron boundaries from the DNA

sequences of mouse cDNAs prepared by RT-PCR and the mouse gene sequence. Total RNA

was prepared from mouse mammary tissue (day 1 of lactation) (Chomczynski and Sacchi,

Anal. Biochem. 162: 156-159(1987)) and reverse transcribed into cDNA by incubation with

MuMLV reverse transcriptase and random hexamers at 42° C for 15 min, following the

protocol described in the Perkin Elmer RT-PCR kit (Perkin Elmer Corp. , Branchburg, NJ). The cDNAs, cDNA 1, 2, 3 and 4 (FIG. IC) were then prepared by amplifying the indicated

regions of DNA by the PCR. The amino acid sequence was predicted from the verified

cDNA sequence using the TRANSLATE program from the Wisconsin Genetics Computer

Group (GCG) (SEQ ID NO:4). Based on this amino acid sequence, the translational initiation

codon, AUG, is predicted to be at nucleotides 4923-4925 of SEQ ID NO: 1. This site is

consistent with the predicted location of translation initiation on bovine butyrophilin mRNA

and is also within the preferred context for most eukaryotic genes (Kozak, Nucl. Acids Res.

15: 8125-8248 (1987)). There are four other potential AUG initiation codons at positions

4650,4743,4765,4776 of SEQ ID NO:l between the most distal transcriptional initiation site

at position 4611 and the predicted translational start site at position 4923 (SEQ ID NO: 1).

However, the most distal of these AUG codons is not within the preferred sequence context and the other three are almost immediately followed, in-frame, by the stop codons TAA, TGA

and TAG, respectively. In almost all such latter cases the RNA polymerase continues to scan

the mRNA for the next potential AUG start site (Kozak, Nucl. Acids Res. 12:3873-3893

(1984)).

Comparison of the DNA sequence of Btn with that of butyrophilin cDNA also revealed

that, like many other genes in the IgSF (Williams and Barclay, 1988), there is a close correlation between exon organization and functional units of the protein. Thus, exon 1

encodes all of the 5'-UTR and the signal sequence; the location of the signal sequence is

designated by the vertical dashed line in FIG. IB. Exons 2 and 3 encode the I-set and C-set

immunoglobulin-like domains, respectively, and exon 4 encodes the membrane anchor.

Tissue Specific Expression of Mouse Butyrophilin. Previous work has suggested that

butyrophilin is specifically expressed in mammary tissue and that expression is maximal during

lactation (reviewed in Mather and Jack, 1993). However, this conclusion was based on the use

of either relatively insensitive protein and RNA blotting techniques, or immunofluorescence

microscopy. Thus, the expression of mRNA in mouse tissues was analyzed with a much more

sensitive RNase protection assay.

Riboprobes were prepared from a mouse cDNA, mcDNA3 (FIG. IC), subcloned into

pCR II (Melton et al. , Nucl. Acids Res. 12:7035-7056 (1984)). For anti-sense riboprobe, the

plasmid was linearized by digestion with Xbal and the RNA synthesized using SP6 RNA

polymerase. For sense riboprobe, the plasmid was linearized by digestion with Hindlll and

the RNA synthesized using T7 RNA polymerase. In each case the RNA was labelled by the

inclusion of [α-³ P]-dUTP (> 800 Ci/mmol) in the reaction mixtures. Total RNA was prepared

(Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987) from 13 tissues (pancreas, intestine, spleen, liver, kidney, heart, lung, uterus, ovary, thymus, brain, salivary gland, and

mammary) of three Balb/c mice at day 1 of lactation and mammary tissue was pooled from

three Balb/c mice at each of several developmental stages (pregnancy, lactation, and

involution). Anti-sense or sense riboprobes (2 x 10⁶ cpm/sample) were incubated overnight

at 47° C with 10 μg total RNA in 30 μl of a hybridization solution (80% (v/v) formamide, 1

mM EDTA, 10 mM sodium citrate and 300 mM sodium acetate, pH 6.4 (Ambion, Austin,

TX)). The RNA in each sample was then digested at 37 C, for 30 min with RNAse One

(Promega) (5 U/sample) according to the manufacturer's instructions. RNA was recovered

following standard procedures (Sambrook et al, 1989) and the samples separated by

electrophoresis in a 6% (w/v) denaturing polyacrylamide gel. Radiolabelled riboprobe

protected from RNase digestion was detected by exposure of the dried gel to X-ray film.

The size of the anti-sense riboprobe was such that hybridization to butyrophilin mRNA

was expected to protect a 625 bp RNA fragment from digestion with RNase. A radiolabelled

fragment of the predicted size was only detected in mammary tissue, out of the total of 13

tissues analyzed (data not shown). Analysis of mammary tissue at different developmental stages showed that butyrophilin mRNA is detectable during pregnancy, lactation and involution

but not in glands from virgin animals (data not shown). Expression of butyrophilin mRNA

appears to increase markedly in the last half of pregnancy and remains at relatively high levels

throughout lactation. Analysis of the Btn Promoter. Because Btn is specifically expressed in the mammary

gland and is associated with the MHC or MHC-related genes (Vernet et al, 1993; Amadou

et al, Genomics 26: 9-20 (1995)), a search for similarities between the Btn promoter and the

regulatory elements of mammary -specific or immune system genes was conducted. Approximately 1.8 kb of Btn 5' flanking sequence, shown in SEQ ID NO:2, was analyzed on

either strand by comparison with sequences in the Transcription Factor Data Base (TFD)

(Faisst and Meyer, Nucl. Acids Res. 20: 3-26 (1992)) and by comparison with the published sequences of the whey-acidic protein, α-lactalbumin, β-lactoglobulin, and casein genes.

Over thirty different classes of potential regulatory elements were identified throughout

the sequence. Elements within Btn previously shown to be functional in the promoters of other

mammary-specific or immune system genes are indicated in FIG. 2. For the sake of clarity,

other elements are omitted, unless they are specifically discussed further below.

The mammary-related factors include three potential STAT binding sites identified

using the general STAT consensus 5'-TTNC(N)₃AA-5' (Ihle _an(ι Kerr, Trends Genet. 11 :69-74

(1995) herein incorporated by reference) (asterisks, FIG. 2). Additional STAT binding sites

can be identified using a broader consensus, TT(N)₅AA, based on the work of Lamb et al.

(Nucl. Acids Res. 23: 3283-3289 (1995), herein incorporated by reference) (no asterisks, FIG. 2). Several C/EBP sites were identified, including one between nucleotides -1505 to -1514,

which is the imperfect palindrome 5'-ATTAGGTAAT-3' (SEQ ID NO:5). There appear to

be no sites for the pregnancy-specific mammary nuclear factor (5'-TGAT/ATCA-3\ Lee and

Oka, 1992, herein incorporated by reference) or the single-stranded nucleic acid binding proteins (various consensus sequences checked, see Altiok and Groner, 1994, herein

incorporated by reference). Btn contains potential binding sites for NF1, Ets-related proteins

(PU. l site, Klemsz et al, 1990, herein incorporated by reference), heptamer binding sites for

Oct 2A, which will bind Oct 1 (Kemler et al , EMBO J. 8: 2001-2008 (1989), herein

incorporated by reference) and glucocorticoid response elements (^■/^•> sites). There are several

YYI sites and at least 11 GMCSF elements (Nimer et al, 1990, herein incorporated by reference) which will also bind YYI (Ye et al. , Nucl. Acids Res. 22: 5672-5678 (1994), herein

incorporated by reference). Two negative regulatory elements characterized in the whey-acidic

protein gene promoter were identified (Kolb et al , 1994, herein incorporated by reference).

These elements (allowing one mis-match each) are within the appropriate context in Btn,

approximately 270 nucleotides apart in the proximal region of the promoter. Most

significantly, no "milk-box" region was found using the consensus sequence of Laird et al.

(1988), herein incorporated by reference, and no obvious CoREs with composite C/EBP,

glucocorticoid response elements and STAT5 sites (Raught et al , 1995, herein incorporated

by reference) were identified. Furthermore, comparison of the 5' flanking region of Btn with

promoters of the casein, whey acidic protein, α-lactalbumin and β-lactoglobulin genes by

FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448 (1988) or BLAST (Altschul et al , J. Mol. Biol. 215: 403-410 (1990)) showed only limited similarities. The Btn

promoter therefore appears to have novel features with respect to the regulatory elements of

other mammary-specific genes.

The Btn promoter lacks the characteristic response elements associated with classical

MHC class I and class II genes (Le Boutellier, 1994; Dorn et al , Proc. Natl. Acad. Sci.

U.S.A. 84: 6249-6253 (1987)). However, there are many potential regulatory elements

associated with immune system genes including α- and γ-interferon response elements and

consensus sequences for TCF-1 (Faisst and Meyer, 1992, herein incorporated by reference)

(not shown in Fig. 2), and PU. l (Klemsz et al, 1990). A repeat element of three GMCSF

sites in the proximal promoter was identified which in the same context has been shown to

regulate the mitogen-inducible expression of GMCSF in T cells (Nimer et al, 1990). Accordingly, the mouse Btn promoter may be used to direct the expression of desirable

proteins in the milk of transgenic animals and to screen for compounds that are mitogenic. As

used herein, the term mouse Btn promoter means all the sequenced nucleotides from 1 to 4693

of SEQ ID NO: 1 or a substantial equivalent. A substantial equivalent is defined as a DNA

sequence which enables a DNA fragment containing this sequence to hybridize under stringent

conditions to a DNA fragment containing nucleotides 1 to 4693 of SEQ ID NO:l.

In addition to the regulatory elements found in the promoter region of a gene, there is

evidence that regulatory sequences involved in tissue-specific expression may also be located

in the transcriptional unit of the gene or in 3' flanking sequences (See, e.g. , Charnay et al ,

Cell 38:251-263 (1984); Gilles et al. Cell 33:717-728 (1983)). Thus, the cloned butyrophilin

gene may be used as a source of such regulatory sequences. For example, a rDNA construct

for expressing a heterologous protein may include a DNA sequence coding for the protein inserted into the first exon of the Btn gene. Preferably, the insert is precisely fused to the Btn

signal sequence for targeting the heterologous protein into the secretory pathway normally

involved in secreting butyrophilin into milk.

Example 4: Cloning and Analysis of the Bovine Butyrophilin Promoter

The 5' untranscribed region of the bovine butyrophilin gene may be cloned from bovine

genomic λ phage libraries by standard hybridization methods using the bovine butyrophilin

cDNA disclosed in Jack and Mather (1990). By sequencing a clone containing the bovine

promoter, herein referred to as BTN1, and comparing the sequence with the mouse promoter sequence, the boundaries of the bovine promoter and regulatory elements contained therein

may be identified. Example 5: Preparation of Synthetic Butyrophilin Promoter Regions

It will be understood by those skilled in the art that an entire butyrophilin promoter may

not be necessary to provide a desired biological activity. For example, if production of a

heterologous protein in milk is desired, there will be some minimal region or combination of

regions within a butyrophilin promoter that is necessary and sufficient to respond to the

transcription factors that control expression of the butyrophilin gene in lactating mammary

tissue. On the other hand, if the object is to screen for compounds that are mitogenic, there

will be some minimal region or combination of regions in a butyrophilin promoter that are

necessary and sufficient to direct expression of butyrophilin or other gene in the presence of

mitogens. Such minimal promoter regions that are necessary and sufficient to provide a desired

biological activity may be identified by deletion analysis using methods well known in the art.

In brief, deletion constructs are prepared containing increasingly smaller portions of

λBtnl or BTN1 operably linked to a reporter gene (e.g. , see Example 6 below) and the

amounts of reporter gene expression in response to various transcription factors are compared

among the deletion constructs. The minimal promoter region(s) of λBtnl or BTN1 which

provide the desired response are then either subcloned from the deletion constructs or

constructed from oligonucleotides synthesized on an automated DNA synthesizer. It will be

understood that these minimal regions may comprise DNA sequences derived from a

butyrophilin gene or their substantial equivalents, as defined above. These minimal promoter

regions may then be operably linked to a desired coding sequence and placed in a recombinant

expression vector. Example 6 - Construction of Butyrophilin:hGH Expression Vector

The Allegro^® HGH Transient Gene Expression Immunoassay System (Nichols Institute

Diagnostics, San Juan Capistrano, CA) may be used to evaluate butyrophilin promoters or

promoter regions. In brief, a DNA fragment containing a butyrophilin promoter or minimal

promoter region is cloned into the pøGH vector which contains the human growth hormone

(hGH) structural gene but lacks a eukaryotic promoter. The resulting fusion plasmid is

transfected into a primary mammary cell line, and the hGH secreted into the medium is

detected immunologically using a monoclonal antibody -based assay (Nichols Institute Diagnostics, #40-2205). Since the level of secreted hGH is proportional to mRNA levels,

promoter activity can be monitored.

In addition to hGH, other reporter genes such as those encoding chloramphenicol

acetyltransferase (CAT), green fluorescent proteins or luciferase could be used to evaluate

butyrophilin promoter regions. Detection of the products of these reporter genes products is well-known in the art.

Example 7 - Construction of Transgenic Animals

The production of transgenic mammals containing a foreign DNA sequence coding for

a desired protein or polypeptide in its germ line is accomplished by procedures well-known in the art. For example, see Rosen, U.S. Patent 5,304,489 (transgenic mice) and Clark et al. ,

U.S. Patent 5,322,775 (transgenic sheep), each of which is herein incorporated by reference.

Generally, the process comprises collection of embryos, injection of the DNA into the

embryos, transfer of the surviving embryos to surrogate mothers, and screening the offspring

for integration and expression of the foreign gene. To construct the transgenic animals

embraced by the invention, the injected DNA would be a rDNA construct comprising a butyrophilin promoter or minimal butyrophilin promoter region(s) operatively linked to a DNA

sequence encoding a desired polypeptide. The DNA construct preferably also comprises a signal sequence operatively linked to the DNA sequence encoding the desired polypeptide.

In addition to constructing germ-line transgenic mammals, the invention contemplates

the expression of desired coding sequences under the control of a butyrophilin promoter or

promoter region(s) in somatic transgenic mammals. As described by Lothar Hennighausen,

J. Cell. Biochem. , 49: 325-332 (1992), herein incorporated by reference, such animals may

be generated by the physical introduction of DNA with a jet injection gun into the mammary

epithelial cells of a living lactating animal. See also Furth, P.A. et al. , Gene transfer by jet

injection into differentiated tissues of living animals and in organ culture, Mol. Biotechnol. ,

4(2): 121-127 (Oct. 1995), herein incorporated by reference.

Example 8 - Detection of Disease States Associated With Expression of Butyrophilin in

Nonlactating Mammals

As discussed above, butyrophilin is a member of the IgSF and its cytoplasmic domain

is similar to the cytoplasmic domains in zine-finger proteins. Thus, the expression of

butyrophilin in RNA in nonmammary tissue or in mammary tissue of nonlactating animals may

be useful for detecting cancer and other disease states in which the butyrophilin promoter is

activated.

The principles, preferred embodiments and modes of operation of the present invention

have been described in the foregoing specification. The invention which is intended to be

protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than restrictive. Variations and changes

may be made by those skilled in the art without departing from the spirit of the invention. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: MATHER Ph.D., IAN H. OGG Ph. D . , SHERRY L . JACK Ph.D., LUCINDA J.W. KOMARAGIRI Ph.D., MADHAV V.S.

(XX) TITLE OF INVENTION: THE BUTYROPHILIN GENE PROMOTER AND USES THEREOF

(xxx) NUMBER OF SEQUENCES: 5

(xv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: WATSON COLE STEVENS DAVIS, P.L.L.C.

(B) STREET: 1400 K. STREET, N.W.

(C) CITY WASHINGTON (D) STATE: D.C.

(E) COUNTRY: USA

(F) ZIP: 20005-2477

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: POULOS III, JAMES A.

(B) REGISTRATION NUMBER: 31714 (C) REFERENCE/DOCKET NUMBER: 6067/JAP69170A

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-628-0088

(B) TELEFAX: 202-628-8034

(2) INFORMATION FOR SEQ ID NO : 1 : (x) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14180 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI -SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(vii) IMMEDIATE SOURCE: (A) LIBRARY: 129 ES cell genomic library

(B) CLONE: Lambda Btnl

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: 13

(ix) FEATURE: (A) NAME/KEY: TATA_signal

(B) LOCATION: 4645..4651

( ix) FEATURE :

(A) NAME/KEY: misc_feature

(B) LOCATION: 4611 (C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "transcription start site"

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 4675

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence--^* EXPERIMENTAL /s andard_name= "transcription start site"

(ix) FEATURE: (A) NAME/KEY: misc_feature

(B) LOCATION: 4694

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence*-- EXPERIMENTAL /standard_name= "transcription start site" (ix) FEATURE:

(A) NAME/KEY: polyA_signal

(B) LOCATION: 13091..13096

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence*^*- EXPERIMENTAL (ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 13097..13199

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "3' end of transcript"

(ix) FEATURE:

(A) NAME/KEY: misc_signal

(B) LOCATION: 4923.-4925

(C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /evidences EXPERIMENTAL

/standard_name= "Translational initiation codon" (ix) FEATURE:

(A) NAME/KEY: misc_signal

(B) LOCATION: 4650..4651

(C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /evidence= EXPERIMENTAL

/standard_name= "Translational initiation codon" /pseudo

( XX) FEATURE :

(A) NAME/KEY: misc_signal (B) LOCATION: 4743..4745

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name-= "Translational initiation codon" /pseudo (xx) FEATURE:

(A) NAME/KEY: misc_sιgnal

(B) LOCATION: 4765..4767

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "Translational initiation codon"

/pseudo

(ix) FEATURE:

(A) NAME/KEY: misc_signal

(B) LOCATION: 4776..4778 (C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "Translational initiation codon" /pseudo

(xx) FEATURE: (A) NAME/KEY: intron

(B) LOCATION: 5002..5520

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "Intron A" (ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 5872..8332

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "Intron B"

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 8615..9485

(C) IDENTIFICATION METHOD: experimental (D) OTHER INFORMATION: /evidence--^* EXPERIMENTAL

/standard_name= "Intron C"

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 9636..10206 (C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence= EXPERIMENTAL /standard_name= "Intron D"

( ix) FEATURE : (A) NAME/KEY: intron

(B) LOCATION: 10228..10320

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence-- EXPERIMENTAL /standard_name= "Intron E" (ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 10348..10738

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /evidence-- EXPERIMENTAL /standard_name= "Intron E"

(ix) FEATURE:

(A) NAME/KEY: mιsc_feature

(B) LOCATION: 4807..4814

(D) OTHER INFORMATION: /standard_name= "Inverted repeat" (ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 12556..12563

(D) OTHER INFORMATION: /standard_name= "Inverted repeat"

(ix) FEATURE: (A) NAME/KEY: promoter

(B) LOCATION: 1..4693

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 4923..5001 (ix) FEATURE:

(A) NAME/KEY: misc_signal

(B) LOCATION: 11395..11397

(D) OTHER INFORMATION: /standard_name= "Translational stop site"

Cxi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

AAGAATAAGA CACACACACA CACACACACA CACACACACA CACACACAGA GAGAGAGACA 60

GAGACAGAGA CAGAGACAGA GAGAGACAGA GAGTAAAATG CATCCAAAAT ATAAATGGTT 120

ATATAGAGAA ATAAAAGCAT AAATGAGTTA TTGTGTTCAG AAACGTGTAG ACAAGTAACA 180 GATAAAGCAG TTTTACATAG ACTACCACAG TGGCTCATGA CATTAATTTC AGTATTCAAG 240

AGACTGAGGC AGGAGGATCA CCATGGCTTC AAGACCATTC TAGACTATAT AGTAAGTTTC 300

AGGCAAGCCT GGGGTACCTT GCCTCAAAAT AAATAAACAA ATAAAATTAA AAAAAAAAAA 360

AGTAATGAGC AGCCATCTTG GCTTCACCTA CCTATAATCC CAGCCTTGGG AGGTGGAGGC 420

AGGAGGATAA TTGCAGTGAT ATCTTCAGCT GTATAGTGTT CTTGGTGCCA CCCTGGGCAA 480 TAAGATACAG TTCTCCACCC TCCTACAAAA GTACAGTTAT ACTTGTTTGC TTTTGAAAGA 540

AGCTATGGAA GTTACCACCC TCAGGTGACT TTTGAGAGAG GGAGGGGAAT TAACCATGCA 600

GACAAGACGG GGCAGAATGT TCTGGGAGAG ACGATGAGCA CCATTATCTG GAGGAGGTGC 660

TTTGAGTAAC CACACCAATT CCGAGTTTGG CCTGCTAGTG GGACAGTGCA GGAAGAGGAA 720

AGAGAAAGGC TTTTCCTTTT CTTCAATCTG TTACCATGGA AACATCTTTG TCATCTACAA 780 AGAACACATT GGAGGAAGGA AAAGAAAAAA AAAAAAACAA ACACAACATG ATCTGTGAAT 840

GAGTCTGTGT TGAGTCTCAT TCAGAGTCAC CCTGGAGAGA TGTGTTACAT GCGGCTGTGG 900

GTCACAGGTT GAACATGCCC AGGACTACCA CAGTGCTTGT CCCTCCCTCC CTTCCAGCTG 960

TCTTTCTCCT GTTTTTATTT TGAGACACAG TGTCCCTGTG TAGCCTAGCT GGTCTGTGTC 1020

TTGTTTTGTA GGCCAGGAAC CCTCCCCCCA CCCCCAACAC ACACACACAC AAACTCAGAG 1080 CAATGCTCCT GCATCAGCCT CCTGTCTGTT ACAAATAGTC ATCTTAATTA CATGTCTTCC 1140

TAGAGCCTAA GGGTTCTGAC GTCAGTGTGG TTCCAAGTCC CAAGTGATGA CAAAAGCCCA 1200

TCTTAAATTA TTCAGTAATC AGTAATATAT TTAAAGATTT ATTATTAGTA GTGTTAGTAT 1260

TAGTAGCAGT ATTTGGTTTT TCGAGATAGG GTTTCTCTGT GTATCCCTGG CTGTCCTGGA 1320

ACTCACTCTG TAGACCAGGC TGGCCTTGAA CTCAGAAATC CGCCTGCCTC TGCCTCCCAA 1380 GTGCTGGGAT TAAAGGGGTG CACCACCACG GCCTTATTTT TCTTATTTTT CATTTGTGTG 1440

TCTGTGTGTG TGCTCAATTG TGGGTTTGTG CACACAAGGG CAAATGCTCC AGCAGTCCAA 1500

AGAGTGTCGG ACCCCTGGGA GCTGGCATCC CAGGTGGCCA TGAACCACTT GACATAGGTT 1560

TCTCTACAAG AGCAGCCATG TTCTTAACTG CTGAAACATC TCTTCAACCC AACTGTTAAT 1620 ATTTTTGTTC TTCACTCAAA TAAGCTAGGA TGGAACATTT AAATGTATTG TATACGTCAT 1680

TTTAAAATAC AAATTGCCCA CAACTGATGA GGCAAGAGTT CGGAGTAAAG TTCTGAAACT 1740

GCTATCTTGA TAATATGCAT TTCCTGTAGG TATGAAGGAG ATGAGTGTGG CATTTCTGGA 1800

TAGCATTCAG ATACACAGGG ATTGTTACAT TCTCAGTCCT CATGCCAGTC CTCAGCATGC 1860 AGAAAATATT AAGCAAAGAA GTAATAAAAT CAGATGTGTG CTTGGGAACA GCACACAACC 1920

TAGCAGCCAA TCAGGCCAAC GAGAGTAGCT AGCTTTGTTC TTAATCATAG TAATTTTTAA 1980

AAATTAAATA AAATTGAGAA GAAATGTTGT CAAAAATATA AAGCACTTAC TTTAAAATTT 2040

GTTTTTAATT TATTTTTCAT TTATGTGGAT GCATGTTAGT CTGTCATGTG TGTGTGGATA 2100

CCTGGGAGGC CCCTGGAATT TTAATTACGG ACCATGTGGG AGCTGGGAGC TGAGCCCGGG 2160 CCCTCTGCAA AAGCAGCCAG CGCTCTTAAC TTCTAAGCCA TCTCCTCAGA CTTCAAATAT 2220

AATACAATTA TTAACTTTAA TTTTTAAAAA GTCCACACAG AAAGAAGACC AGACCTCAAA 2280

ATAGACAGCG ACTGGTCTGG AGGACTCCAG TCTAGATTTT ACCGAGTGGT CAGCTAATCC 2340

AAGAGAAATG CCCAGCCTTG TTACACCACA AAGGTGATAA TGATGATACT AAATTTCACT 2400

AATTTTCATA TAAGCATGTA AGATAATACC TCTTGTTGCT TGCAGGCCTG AAGGACACTC 2460 TTTAGGATGC TATGATCTTT TTAATATTGT AGGGAAGGTC ACTGATAACA TATATATTTA 2520

TGCCCTTTGA GTCAATGGCT TTATTCATGG AACTGGATCA AACAGCATAT CGGGTAGTTA 2580

TCATAGTTAG AAACAAAGAG CTACAAATAA AAATGCATAT CTTTTTTCTA TTTTCTTCTT 2640

CTTCCTCCCT CCTCCTCTTC CTCCTTTTCT TTCTTGTTTC TTCCTTTTTT TTTTTTTTTC 2700

CCAGGCAGGC TCTCACTGCA TTGCTCTAGT TGTCCAGGAA CTTGATCTGT AGATCAGGCT 2760 GGCCTCAAAC TCTTCCTACC TCTGCCTCCC GAATGCTAGG AATAGAAGCA TGTGTTAGCA 2820

TGTCTGTTTT ACTGTGCGTT TAAAAAAAAA AAGTTTATCT TGCCCTTACT GTTTGCTACA 2880

GGCTAGTAAA ACAAACAGAC ATGGTAGATC GATCTATCTG AGTTACAAAA ACAGACCTTC 2940

TTCGAGCCGG ATTCGAACCA GCGACCTAAG GATTTCCAGG TCGAATACTC CTACAGTCCT 3000

CCGCTCTACC AACTGAGCTA TCGAAGGATA CCATGTATAG TGCCTAGCAA AGTCACAAGT 3060 AGCTTAGAGG AGCCACTATG CCTGATTTTA AGCAGTGCTG GGATCTAACT CAGGGCTTCA 3120

TGAATGCTAG ATGGACCTTC TACCAAATGC CAAGTGCATT CTTTTTTTTT TTTTTTTTAA 3180

TTAGGTAATT TCCTCATTTA CATTTCCAAT GCTATCCCAA AAGTCCTCCA TACCCTCCCC 3240

CCGAAGTGCA GTCTTTATAC TAGAAAAAGA ACTAGAAATC TCATAATCTT CGCAAATATA 3300 TGCGTATTAG CTATGCTATG AACTATGCAG GAAAACTTAC TATGAACTTA TCACTATGAA 3360

CTGATATATA TTGTTCTTAA ATTTTATTTT ATATTTATGT ACAGCATAGA AACAATCATT 3420

GATAAAACTG TTTTTTTTCT TTATCTTTGC ATTTTTTCAG TAATAAATGA AAATTCAAAA 3480

CCAAATAAGA AATTGCTGAT CTCATGACTG ATGGCAGGGT GAAGCGCCAG GTCCTTGTGC 3540 AGTTATACCT TGAAGGTGGA CATCCAGTGG ACTCCTGCCA CCCACACCCA CATTCCTGAA 3600

GGTGTCTCAT GGAAAAGATC AGGGAGGGAG AGCTGCAGCC ATTGTGGACT CACTCTTTAG 3660

CTATTCACAG ATGTAATGAC AAAGTAATTT ACTTTCTGGG CTCCTATTCT CTTGCCTGTT 3720

TTGTTTCCAA TACTGTTTGT GTCTAATACT TTTCCAACTT GGCATAATTC AAACAAGGTA 3780

TTAGTAACAT TAGTCTTTTT CTTAAAAGTA ACAAACACCC CACTCTCTTT TGTTTTGTTC 3840 TCCATATGTA GCTCTTGCAA GTCTGGATCT TGCTATGAAG CCCAGACTGG CCTTAAACTT 3900

ACAATGACCC CCGCCTGCGC CCCCCCCTCC CCCCCCATGA ACTTGGGTTA AAAGAACTGA 3960

AGCCACAGAG TTAAATTCAC AGGCTGATGG CCTCATGACT CATTTCAGTT GCTCAAGTCT 4020

TCTTTCTTTT TGTCCCCATT CCCTATATTC GGTACAGCTC TTTAATGCAT ATATCGTTCT 4080

CTTAGGGGAG GAGGATGAAC CCAAACTACC TGACCACTAA TCTGTAGTCC ACATGTTTAA 4140 AAGGCTGCTC CTCCCCCCAC CCCGAATAAA TACACTTGGT CACCTGTGGG CAGGCTTCTC 4200

TAACAGCACA CAGCCTTCTT CCTTCTGAAG AGCTCTCTCT TTGGCCCCGG GGTGACAAGC 4260

AGCCCTTTTC ACTTGATCAC TGTGGCTCTG GCTCCCTTTT CCTCTGGGTC TGTCGAAATC 4320

GGTAGGTGCT TCACTCTCAG CTCAGCTCTC TTTGTCTCTT CTCTGTACTA GGCTTTCTGT 4380

TCCTCAAGCT CTTCAGCTCT GCCTCTCCCC TCTCTCTCAG ACTTTGTCAA GACTGTATGT 4440 ACCTCACGGT GTAACTCCCA GAGATCACCC TCCTGAGAGC TGCTGGGCTT ACAGTTGAGA 4500

AACACACCTT GTCTTTCTCT CCTCCTTCGT TTCATTTCAT GTTCTCCATT TCTACCTCCG 4560

TGGCTTTATC TTCATTATCA CTTCTAAACA CGAATAACAA AGTATCCCAC TCGATTCGAT 4620

TTTACTTTAT TGTTTTATTG TTATTGTAAA TGAGGAGATT TCTTCATTAT CTACAACTGT 4680

GCCTCGCGGC TCCATTCTGG AGGCAGTCGA GGGCTGGAGG ACCAGACGTA CAGAGGAAGG 4740 GTATGGGGCA GGCGCTGTTG TAAAATGGAC TGAAAATGAC CCTGTAGGGG AAATACAGAG 4800

CCCTCCAGGT TGGAAGAAAC TGGTGGAGAA CAGGGCGCTT GCGGAACCCA TAGTTACCTC 4860

CTGACTGTTT CTCTCCCAGC CTGAAGCTCT TGGCGGGCTT CATTGCCCCA GTTAGCTCAG 4920

AGATGGCAGT TCCCACCAAC TCCTGCCTCC TGGTCTGTCT GCTCACCCTC ACTGTCCTAC 4980 AGCTGCCCAC GCTGGATTCG GGTAAGTTTC TGTTCTAGCC TTCTCTTTCT CGCAAAGTTG 5040

GAAGGTCCCT ATAAATAAAT ACCTCTGACC CGGTTTGGCT CCTGGTGGGG GGACCTTCAC 5100

CACAATCCAG TGAGTTCAAA GGAAACCCAC TGCGGGAGGT AATACACACC TGCAATTGCA 5160

GCACCAGGTG GGCCCAGGCA GATTTTTCTG AGGTCAAAAC CTGCCTGGTC TACATAGAGA 5220 TAGCCAGAGC ATCCAGGGTT ACGTAGGGAG CGCCTAGTTG TTTTTCCTTT AAATCAAAGG 5280

AATTGGAACG CTAAGTGTGG TGGTGGTGCA CCCCTGTAAT GATCGCACTT GAGAATTGAG 5340

GGCAAGGAGC TCAAGGCTAC ATAGTGAGCT GGAGGCCACC TTGGGATTTA TGAGATCCAG 5400

TCTGAAAAAT AAACAGAAGA AAAGAAATAG CAGCCACCCC GAGTTCCTTT CTTTACAAGG 5460

AGACTGGCCG GTAGGTCCTC CATCCCAACC CATCGTCCTA TCTGACCTTG TTTATTACAG 5520 CAGCTCCCTT CGATGTGACC GCACCTCAGG AGCCAGTGTT GGCCCTAGTG GGCTCAGATG 5580

CCGAGCTGAC CTGTGGCTTT TCCCCAAACG CGAGCTCAGA ATACATGGAG CTGCTGTGGT 5640

TTCGACAGAC GAGGTCGACA GCGGTACTTC TATACCGGGA TGGCCAGGAG CAGGAGGGCC 5700

AGCAGATGAC GGAGTACCGC GGGAGGGCGA CGCTGGCGAC AGCCGGGCTT CTAGACGGCC 5760

GCGCTACTCT GCTGATCCGA GATGTCAGGG TCTCAGACCA GGGGGAGTAC CGGTGCCTTT 5820 TCAAAGACAA CGACGACTTC GAGGAGGCCG CCGTATACCT CAAAGTGGCT GGTGGGTACA 5880

GACGGGATGT GTCGCCTCGT CACTCCGCGC GGAGACTCTC ACTTTGGGGA GAATCATCGT 5940

GTTCATTCTC CAAATCCAAA CGTATTTTCA CGTTTACGTA AGGTTGTGGT GAGCATCTTA 6000

GATGCTCTGA ACAGCTTCGT GGTTTAATGC CTAAGGATTG ACACCCTAAC AGAGTGTGGT 6060

CCGTTGCTAA AGTTCTTTAT CCACCTCCAA AATGGTTTTA CTCATATTAC TCATGTTGTC 6120 TTCTTCTCCC TGTCTGAGAT CATAAGGAAA GAATACATTG AGCTCTAATT TCCCTCCCTG 6180

TTAGTGATCC AAATCAAGCA AATCTCCCAC TCAGTTTTTC CTACTGTGAA ACCAGAAAGC 6240

TAAATCCAGC AAGAATTTGC AACAAGGAAC TAGATAAGTG AAAAATGCTT TGTTAATGAT 6300

AAAACATCAT GTGCTTATAA AGAAATTCCT ACACCTTAGA CTACTGTGTA TAATACACAT 6360

ATTGCCTTTC TCATTTATTT AGGTATTTTC CTTGCTCCGT TAAGAAAGGA GCTGACATAG 6420 TGTCTCAAAC TCTACAGCTT TAAGAACACT TTGAAGTCCT TTATCAAGTA CTAGGATCAT 6480

TCGTAAAACA ATGAGTTTCC CACACCGGGA GTCGAACCCG GGCCGCCTGG GTGAAAACCA 6540

GGAATCCTAA CCGCTAGACC ATGTGGGAAC TGCTATGCAT ACTTATCTTG CCTCCTCCTC 6600

CCATGTAAGG ATTCCGGACG ATGACACACC TGCTCTTTAG ATGTTGGGAA AGGAATCTAT 6660 CAACTTAACT GTATCCCTAG CTCAAAAATA CATTGCCATG TTTTGCCATA TTTAATGTAC 6720

CAAATATAAC GCTCATATCA TTTTTAGGGA AAGGCATCCT AAAATTATAT AATATATAAA 6780

TTATATAATA TATACATATA CATGAAAATA TGTGTATATA CATATATGTA CATAAATATA 6840

TGTTTATATT CACATATACT TGTGGGTTTG TGTATGATAT TTCAACTGGG AAGTAACACC 6900 CTGTAATTCC AGCAACTGGG AGATACAAGC AGGAAGATTA GAAGTTCAAA ATTGACCTTG 6960

GCTACTTAGA ACCTCGATGT TGTTATTATC TTTTATAAGT GATGGCCATC TTCATAAAAT 7020

GAGTTTAAAT TTTTCATACA CACTCTTTTG AATATGAAGA GCTGTTGAGG TGTTGTTTAA 7080

GGATACTTTT CTAGAGCTCA GAATTTTTCT GTATCCTGTA GAACTGTGAA AGGGGAGAGG 7140

GGAGAAGGAA AAAGAGTTAG GAAAAGAGGA AAGAGGGAAA TAGAGGGAAG GAAAGATTAG 7200 ACTAAATAAA AATGAAAAGT AGCTTTATGT TACCTTTGTT GCTGCTAATT TTCTGTTGCT 7260

ATTTGTTTGT TTGATTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGCATC AGGGTGTTAC 7320

TATGTAGCTT TGGCTGGCCC CAAACTTGCT ATGCAGACCA GGCTGGCCTA GAATTCATAG 7380

AAAGCCACCT GCTTCACCCT CTCCAGCACT CAGATTAAAG GCCTAGACTA TCACTTTCTC 7440

TGTTGTTATA GAGAAATGGT CTTGAACTCG TTATATAGCA GAGTCTGTTC TAGAACTCCT 7500 GATTCTTCTC CCTCCACCTC CTGAATGCCA TGATTACAGT TGTGTGTCCC CTGTGTTGGT 7560

TTTTGTTGGG GCATAATTCA GTGAGGAAAG ATGAGGTTGA AAACATTTAA GAAAATTCTT 7620

GAGTCTGCAT CCTAGGTAAA GAAAAGTTAA ATTATCAACT GCAAACCTCA AGGGGAAAAA 7680

CAAAACAAAA TTCCAAACTC TGTTCTCACG TATATAGTCT TTTGGGGAGT AGCGGTGTGA 7740

CTCAGTTGGA AGTGTCTTTG CTTAGCATGC ACAAAGCCCT GGGTGGGATC TCTAGCACTG 7800 TCTAAAAATG GTTTGTGGTG GCACGTGTCT CTAAGACCAG CATTTGGGAG GTAGAAGCAA 7860

GAGGATCAGA AGTTCAAGGT CATCTTCGGC TGTTTGAGGT CAGCCTGTGC TACATGATAA 7920

TCTGTCTAAG AAGGAGAATA CTTTCCCACC CATCCTAAAA TATTCTAACC ATAGTCATCT 7980

CATCCTCCAA ATCATGTTAT GCACTTCTAA CCACAGAGGT CTTTCTTGAC TCTAGATCTC 8040

TAAGGCACCT TGCAGCCATT GTTCTTGCTG TTCTTGATAG TTGGACAAAC ACCTCCATGT 8100 CTACTCATCA GACTTTTCCA TATGTTGAAG GATGTGATCT CAATAAGGCT ACATCTCTAA 8160

TAAGATATAA AACATTGTTT TTATTGATCC TTCAGTCTTC ATACAGAAGG ATTAGAGGAA 8220

TCCTACCTAG CCCCAGTCTA CTTTTGCCTC CTCTCTGTCT TTCTCAAGCG AAGATTACCA 8280

TGTTTCCAGG AAAGCATCCA CCAAAAGATT AAGGTCAGTT TCTCTCTAAT AGCTGTGGGT 8340 TCAGATCCTC AAATCAGTAT GACGGTTCAA GAGAATGGAG AAATGGAGCT GGAGTGCACC 8₄00

TCCTCTGGAT GGTACCCAGA GCCTCAGGTG CAGTGGAGAA CAGGCAACAG AGAGATGCTA 8460

CCATCCACGT CAGAGTCCAA GAAGCATAAT GAGGAAGGCC TGTTCACTGT GGCAGTTTCA 8520

ATGATGATCA GAGACAGCTC CATAAAGAAC ATGTCCTGCT GCATCCAGAA TATCCTCCTT 8580 GGCCAGGGGA AGGAAGTAGA GATCTCCTTA CCAGGTCAGT GGAACTAGTG CTGGGTTCTC 8640

ATGATGACAG AGACTCAGGC CAATATGACT TGGGACCCTG CTCAGAAGGG ACATCATGGC 8700

AAAATTGTTT ACATCTTCCC CTACAGCTCT TGCCTGCTGA CTTAAGGAAA TCCTACCAAC 8760

TAAATTAGAA TAAAGATACT TAGGGCTGGG CTGTATCTCT GAGTGCTTGT GTGGCATGCA 8820

GAAGGTCCTA GGTTTTACCC CTTGGTCTGC ACACACCACC TCCATGCCAG TCTCATAAAA 8880 ATTCCAGAGC TTTATTCCAG AGAAACAGGT GATAGAAAAG CTTTGCCTCT GGAGTCCTTC 8940

CTGACAGGAC CCTTCTCCTT CAATAAGCAA GGAGAATAAA TTATTTTTTC TTCTGATTTG 9000

ACTGTACCCT CTCTGAACAT TTCCTCCCTT CCTTGTTCCA CAATGGAGCT CCATATAGGC 9060

CGCCAAAGAC TGCCAAGTTC CTCCAGGAAC TTTCATCATT TCCAATTTAT TACCTGTGAT 9120

TTAGCAGGAA TCATTCCTTG TTTATTGGCC AATGATTTCC ATCCTATCTT GCATGCAATC 9180 ACCTTTCCTC TTCCTTCCCT ACCTCAGCTA CCTCACTGAT AGTTAACAAG GGATTGCTGT 9240

AAATTTTTAT TTCACATGTT CTGACCCCAA CTGGCTGTTC AGTGTTTGCT TTGGCTCAGG 9300

GTCAAATCTT TCTGGAAAGC TTAGCCTGGA GGGGCAATTC TTGCTGTAGG CAGTGTGAGG 9360

CCACTGAGAG CACTCCCATG TCTGTTCTCC TTTGGTATCA GGAGAGAAGC TGAAGTTGTT 9420

CATTTTCCCA ACCAATGTCC TTTTCGGTTT GTTGTTTGTT CATTTTGTTT TGTGTTGTGT 9480 TTTAGCTCCC TTCGTGCCAA GGCTGACTCC CTGGATAGTA GCTGTGGCTA TCATCTTACT 9540

GGCCTTAGGA TTTCTCACCA TTGGGTCCAT ATTTTTCACT TGGAAACTAT ACAAGGAAAG 9600

ATCCAGTCTG CGGAAGAAGG AATTTGGCTC TAAAGGTAAG TCACTGTCCC CAAGGGCTTT 9660

GTGTCTCGGC TTCCAGGGAA GGTTGAATTC AGGGCTGTTT GGATGACTTC CAACAGGAAG 9720

ATGCTGGATT TTAAAATTCC GAGGTTGGAA GGAACGATAA ACCTTCAAAA GTCACAGGTA 9780 CCTACCTACT GTGAAGAAAA GTGCACGTGA CCCAGGCAAA GTCAAAATCA CCTGGAACTG 9840

TCACTGTGTA CCTGATATTC TTTCACAGCC CAGCTGTAGG CTCTCTGGCC AGTCTAACTC 9900

TGTTGCCCAG GAAGAATGTT CTTATTAAGA TCTAGCCCTG AGTCCTAAGC CAGGAGGAAC 9960

TTCCAGGTGA TTTCTTAGAA ATATTCCGGG GAGTCTCTTG TTAATTAATT AATTTATTTA 10020 ATATTTACAT TTTAGTTTAT TTTGTTTTGC TGGCAGCA.TT TCTGTTCCTG GTTTGCAGGC 10080

AGAGTTCCTG TCACCAGGGC ACCACAGAGT AAACAGTGTC CCCTTGTGTG TCCCTCATTC 10140

TGGTTTTCCT CCTTCCCCTT TCCCATTATA AAAAAAGCCA TTGACATAAT TTTGTTTGTT 10200

TTCCAGAGAG ACTTCTGGAA GAACTCAGTA AGTATTTTTG TTTTGTTTTG TTTTGTTTTT 10260 TTGTCACGAG ATTTTCTCTC TCCTACTTGT TAACTGATGG TCTCTTTCCT TGCGTTTCAG 10320

GATGCAAAAA GACTGTACTG CATGAAGGTC AGTGGTTCTG AGCTCCTCAC TGCCTCTGAA 10380

GCCCTTCCGT GGGAGTCAAA GACCTGGGAG GCTTGCACTC CAGACTACCT CCTTAGTAAC 10440

AGGATAGAAA CAGGGAAGGT GACAGCGAAT GGTCTCAGCG CTTTCTGGGA GGCATCGCGA 10500

GGACCACTAG CTAGCAGAAG AGCTCCTTTG AGGGATACCG CATTTGATAG TTCTTAAGTC 10560 ATGCCGTAGC TGCCAGTAAG AGATTGGGGC TAGAGAGAAG GACTGCTAGT GAGTGGCCTG 10620

ATAGCTCCCC TACCACAGCT CCTGCAACTC TATTCCACGT CTCTGGGAAG GGGAGATAAT 10680

TCGGGTAGTC TTGATACGGG GACAGGCTGA TGCAGTCTCT CTTTGCCTCC AGTTGACGTG 10740

ACTCTGGATC CAGACACAGC CCACCCCCAC CTCTTCCTGT ATGAAGATTC AAAGTCAGTT 10800

CGATTGGAAG ATTCACGTCA GATCCTGCCT GATAGACCAG AGAGATTTGA CTCCTGGCCC 10860 TGTGTGTTGG GCCGTGAGAC CTTTACTTCA GGGAGACATT ACTGGGAGGT GGAGGTGGGA 10920

GATAGAACTG ACTGGGCCAT TGGTGTGTGT AGGGAGAATG TGGTGAAGAA AGGGTTTGAC 10980

CCCATGACTC CTGATAATGG GTTCTGGGCT GTGGAGTTGT ATGGAAATGG GTACTGGGCC 11040

CTCACCCCAC TCAGGACCTC TCTCCGATTA GCAGGGCCCC CTCGCAGAGT TGGGGTTTTT 11100

CTGGACTATG ACGCAGGAGA CATTTCCTTC TACAACATGA GTAACGGATC TCTTATCTAT 11160 ACTTTCCCTA GCATCTCTTT CTCTGGCCCC CTCCGTCCCT TCTTTTGTCT GTGGTCCTGT 11220

GGTAAAAAGC CCCTGACCAT CTGTTCAACT GCCAATGGGC CTGAGAAAGT CACAGTCATT 11280

GCTAATGTCC AGGACGACAT TCCCTTGTCC CCGCTGGGGG AAGGCTGTAC TTCTGGAGAC 11340

AAAGACACTC TCCATTCTAA ACTGATCCCG TTCTCACCTA GCCAAGCGGC ACCATAACAA 11400

ATATTCCAGC TTCACGACTT TGCCTTCCTT TGACTAATCC CTCATGCCCC GAAGCTTCAG 11460 CTGTTGGCTT CTTGCAGCCC TGCTTCTTCC TGGTGGATGG AGATTAATTC ACATTGGGAA 11520

GGTTAGGTAT GTTGCTGCCA GACAAGGCAG GAAGAAAGGC CATCCTAGTT TGTTTCTGTA 11580

CTAACAGTGG GGAGGAAGAG AGCTGAATCC TAAACTATTT CCAGTGCTCA TATTCCTTCA 11640

GGCCAGAGCC TATAGAGAAG GATTTGGTAC AATCACTCGA GGGATCAAGA GGCAATTAGG 11700 TTGGCATGGA ATTATGGCAG AAACATCTGG AATAGGGGTA TGTGGAATGA CAGGTTTTAG 11760

GTAAGGGAGA ACAAAACCAA ACCATAGGAT GCTGAGAAAG AAAGATCTTG GACTAAACTC 11820

CTAAAAAAGC ACTTAGAGAA GATATGACAG GCAAATGAAG TGAATTTGGT CTAATTTGAT 11880

ACACTTGCCC TGTCCCTAGG GTTTTTCAGT TATATCTCAA TTTTTTTGTT GTTAATTACA 11940 TTTTTGACAG CTTCATACAT GTATATAATG CATTCTAATT ACTCTCACTC TCCTCTATTC 12000

TGTCTTATTT CCCTCCCCTC CCCTCATACC TTCCTTCTTG CTTCAAACCT GGCACACTGA 12060

GTTTAATGGG CTATCATGGG AACATGGATT TAGAGCTTTC CTCTGAGCTC AAGAGAGCAG 12120

GTGTGACTGA ATACAGTGAT TTCCCCTCTC CTACAATCAA TCAGCAGTCA ATAGCTCAGC 12180

TGGGAGGGGT AGGGCCTCAT GAGACTTCCC CTATCAAGGC TAAATGTTGA AAGGGCCAGT 12240 TTTTAGCACC TGTGAGATCA TGATTGCAAG AGCCCAGAAG ACAGCATTGC TCGGTCATTC 12300

TCCCTACCCT TTGGCTTTTC TGGTCTTTTG TCCTCTCTTT CAGGATGTGT CTGAACTCTG 12360

TATCTTAAGT TTTCTATGTC ATGTTCTATA AGATAGAGGA GACTGGCCCT GCTTGTTTGA 12420

GAGCAATGTG AGCAAGCTAG CAAGAGACAG AAAGGAGCGG AGATGAATAG GGGTAGAGAA 12480

AATTTTTAAA CAAACCCTCC AGGTGTGTGT GTGTGTGTGT GTGTCTTCCT CTTTTTTGAC 12540 CTCCCTAAAG GTCAATCCAA CCTCACATTA TTGACTCCAC TAGGTGGGGG TTCTGTGTGT 12600

GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTTTTAAG ATAGAGGTTT ACTATGTAGC 12660

TTAGGCTGGC TTTGAATTCC TGATCCTCCT GCCTCTACCT TCCAAGTGCT GGAAACATAG 12720

CCACATCCAC CACCCCTATC CAGTCCACCT GGTTTGATTC AGCAACGCTC AGGTAGCATC 12780

GCTGTTTGAT CTGGAGCTGC CAGCTCCCTC GGCCCCCACT GCAATGCTTA ACCCCCTCAC 12840 AGGCACCTTC CCTTGCCTAA CACTGCCATC CTTTTCCACA CTGAGCCATT TGCTCAATGT 12900

AGCCTACCCA GGTATCCTGC TTTCTGGTCC CCAAAGTTAC ACCATGATGC TCAGCACAGC 12960

TGGACAGTTT GTCCCAATTT GTGTGTGTCC TCCTGTTTGT ATGGGACTTC TTTTTGTCAA 13020

TGGCCTGTGT GTGTATCCAA GCTCTTCCAC TTCTATTGTA TTTTTCCGGC TTCTAAAACA 13080

GATGTTACCA AATAAAGAAA GAGAAAGAAA CGAATGTCTG TTTGCTGAAG GCAGCCTCTG 13140 AACTTTTCTT TCTTTATCCC AATAAGAGGG ACTGGATTAA ACCGAAACAG GAATGAGCGC 13200

TGCCTGTCTG GGAAAGTCCT ATTGCAGCAG GGCTGTTCTG TATGGTCCGA GGCTTAGGAC 13260

TGGGAGATTT ACCAGACCAG GCAGAGATGG GAGCTACTCA TGAGGATCAA ACTACCTTCA 13320

AAGAGGCCAC TGTGCTGATG GCTTCCTGCT CTCAGCCTTG TTTCAAAGGC AACTTCATTT 13380 CTATCCCCAC TAAGGTAACT TTGTTGGTGA GTAAACTCCA ACACGGTGCC AGATGTACCA 13440

AGAGGGTGCA GCTCCACAGT AGAGTTCTTG CCCGCCATGC ACCTGCGTGT GTTCCATCCC 13500

TAGCACTGCT TCTGCCCCAC ACATGATTCC TAACAAGTCT CCAAAGACAT GAAAATTGGG 13560

GGATACATTC AAACCACTGC AGGTTCTTTC CTCTATCACC TCATGGGTCC CCGGTGCCCA 13620 GTGTCTTCCT TCTCTTTTTT ATCTCAAACA CTAGCCACCC TATGCAGCTT GTCTTTTACT 13680

GTACTCCTAG GAGAACGGTA TAATTTACCT TTGATTTAAG AGAATTAACT TAATTGAGTG 13740

TGGTGACATG GGCCTGTATT CCCCAGAATT CAGAAGACAG AGGCAAGAGA ATTGTCACAT 13800

ATTTGAAGCC AGCTTGGACT ATATGTCAAT TCAAGGTCAG CCTAAGCTAT ACAGTAACAC 13860

CCTATCTCAT TAAATAAATA AATAAATAAA TGTGTTCATT TTATTCAAAT ATTTTACTTG 13920 TAGAAATCCA CAGAAAATAT AGTCGAAACA TCCTTTCAAA AATTGGTGAG ATGGCTCACC 13980

AGATAAAGAC ACTTACTTGC CAAACCTGAT GACCCGAGTT CAACCCCAGC GACCCACATG 14040

GTGGAGTGAA TTGTCCTCTG ATATCCACAT GTTTGTCATA GATCATGCTC ACCCATACAC 14100

ATATACACAT ACACATGCTA AATATGTTCC ATGTCTAAGA AAGGTAGACT GTTGCATCAC 14160

TGTGTTTAAT GTGTGACAAG 14180

(2) INFORMATION FOR SEQ ID NO : 2 :

^■ (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1750 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI -SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: 129 ES Cell Genomic Library

(B) CLONE: Lambda BTN1 (viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: 13

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

CGAGCCGGAT TCGAACCAGC GACCTAAGGA TTTCCAGGTC GAATACTCCT ACAGTCCTCC 60

GCTCTACCAA CTGAGCTATC GAAGGATACC ATGTATAGTG CCTAGCAAAG TCACAAGTAG 120 CTTAGAGGAG CCACTATGCC TGATTTTAAG CAGTGCTGGG ATCTAACTCA GGGCTTCATG 180

AATGCTAGAT GGACCTTCTA CCAAATGCCA AGTGCATTCT TTTTTTTTTT TTTTTTAATT 240

AGGTAATTTC CTCATTTACA TTTCCAATGC TATCCCAAAA GTCCTCCATA CCCTCCCCCC 300

GAAGTGCAGT CTTTATACTA GAAAAAGAAC TAGAAATCTC ATAATCTTCG CAAATATATG 360

CGTATTAGCT ATGCTATGAA CTATGCAGGA AAACTTACTA TGAACTTATC ACTATGAACT 420 GATATATATT GTTCTTAAAT TTTATTTTAT ATTTATGTAC AGCATAGAAA CAATCATTGA 480

TAAAACTGTT TTTTTTCTTT ATCTTTGCAT TTTTTCAGTA ATAAATGAAA ATTCAAAACC 540

AAATAAGAAA TTGCTGATCT CATGACTGAT GGCAGGGTGA AGCGCCAGGT CCTTGTGCAG 600

TTATACCTTG AAGGTGGACA TCCAGTGGAC TCCTGCCACC CACACCCACA TTCCTGAAGG 660

TGTCTCATGG AAAAGATCAG GGAGGGAGAG CTGCAGCCAT TGTGGACTCA CTCTTTAGCT 720 ATTCACAGAT GTAATGACAA AGTAATTTAC TTTCTGGGCT CCTATTCTCT TGCCTGTTTT 780

GTTTCCAATA CTGTTTGTGT CTAATACTTT TCCAACTTGG CATAATTCAA ACAAGGTATT 840 AGTAACATTA GTCTTTTTCT TAAAAGTAAC AAACACCCCA CTCTCTTTTG TTTTGTTCTC 900

CATATGTAGC TCTTGCAAGT CTGGATCTTG CTATGAAGCC CAGACTGGCC TTAAACTTAC 960

AATGACCCCC GCCTGCGCCC CCCCCTCCCC CCCCATGAAC TTGGGTTAAA AGAACTGAAG 1020

CCACAGAGTT AAATTCACAG GCTGATGGCC TCATGACTCA TTTCAGTTGC TCAAGTCTTC 1080 TTTCTTTTTG TCCCCATTCC CTATATTCGG TACAGCTCTT TAATGCATAT ATCGTTCTCT 1140

TAGGGGAGGA GGATGAACCC AAACTACCTG ACCACTAATC TGTAGTCCAC ATGTTTAAAA 1200

GGCTGCTCCT CCCCCCACCC CGAATAAATA CACTTGGTCA CCTGTGGGCA GGCTTCTCTA 1260

ACAGCACACA GCCTTCTTCC TTCTGAAGAG CTCTCTCTTT GGCCCCGGGG TGACAAGCAG 1320

CCCTTTTCAC TTGATCACTG TGGCTCTGGC TCCCTTTTCC TCTGGGTCTG TCGAAATCGG 1380 TAGGTGCTTC ACTCTCAGCT CAGCTCTCTT TGTCTCTTCT CTGTACTAGG CTTTCTGTTC 1440

CTCAAGCTCT TCAGCTCTGC CTCTCCCCTC TCTCTCAGAC TTTGTCAAGA CTGTATGTAC 1500

CTCACGGTGT AACTCCCAGA GATCACCCTC CTGAGAGCTG CTGGGCTTAC AGTTGAGAAA 1560

CACACCTTGT CTTTCTCTCC TCCTTCGTTT CATTTCATGT TCTCCATTTC TACCTCCGTG 1620

GCTTTATCTT CATTATCACT TCTAAACACG AATAACAAAG TATCCCACTC GATTCGATTT 1680 TACTTTATTG TTTTATTGTT ATTGTAAATG AGGAGATTTC TTCATTATCT ACAACTGTGC 1740

CTCGCGGCTC 1750

(2) INFORMATION FOR SEQ ID NO : 3 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "oligodeoxynucleotide"

(ill) HYPOTHETICAL: NO ( v) ANTI -SENSE: YES

(vi) ORIGINAL SOURCE:

(A) ORGANISM: mus musculus

(vili) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT : 13

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

GGGCTCTGTA TTTCCCCTAC 20

(2) INFORMATION FOR SEQ ID NO: 4:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 524 ammo acids (B) TYPE: ammo acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: protein

(ill) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Mus musculus

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: 129 ES Cell Genomic Library (B) CLONE: Lambda BTN1

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: 13

(ix) FEATURE:

(A) NAME/KEY: Domain (B) LOCATION: 244..270

(D) OTHER INFORMATION: /note= "Membrane anchor domain"

(ix) FEATURE: (A) NAME/KEY: Peptide

(B) LOCATION: 1..26

(D) OTHER INFORMATION: /note= "Signal Peptide"

(ix) FEATURE: (A) NAME/KEY: Domain

(B) LOCATION: 27..143

(D) OTHER INFORMATION: /note= "I-set immunoglobulin-like domain"

(ix) FEATURE: (A) NAME/KEY: Domain

(B) LOCATION: 144..237

(D) OTHER INFORMATION: /note= "C-set Immunoglobulin-like domain"

(ix) FEATURE: (A) NAME/KEY: Region

(B) LOCATION: 304..469

(D) OTHER INFORMATION: /note= "B30.2 Region"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Ala Val Pro Thr Asn Ser Cys Leu Leu Val Cys Leu Leu Thr Leu 1 5 10 15

Thr Val Leu Gin Leu Pro Thr Leu Asp Ser Ala Ala Pro Phe Asp Val 20 25 30

Thr Ala Pro Gin Glu Pro Val Leu Ala Leu Val Gly Ser Asp Ala Glu 35 40 45 Leu Thr Cys Gly Phe Ser Pro Asn Ala Ser Ser Glu Tyr Met Glu Leu 50 55 60

Leu Trp Phe Arg Gin Thr Arg Ser Thr Ala Val Leu Leu Tyr Arg Asp 65 70 75 80

Gly Gin Glu Gin Glu Gly Gin Gin Met Thr Glu Tyr Arg Gly Arg Ala 85 90 95

Thr Leu Ala Thr Ala Gly Leu Leu Asp Gly Arg Ala Thr Leu Leu lie 100 105 110

Arg Asp Val Arg Val Ser Asp Gin Gly Glu Tyr Arg Cys Leu Phe Lys 115 120 125 Asp Asn Asp Asp Phe Glu Glu Ala Ala Val Tyr Leu Lys Val Ala Ala 130 135 140

Val Gly Ser Asp Pro Gin lie Ser Met Thr Val Gin Glu Asn Gly Glu 145 150 155 160

Met Glu Leu Glu Cys Thr Ser Ser Gly Trp Tyr Pro Glu Pro Gin Val 165 170 175 Gin Trp Arg Thr Gly Asn Arg Glu Met Leu Pro Ser Thr Ser Glu Ser 180 185 190

Lys Lys His Asn Glu Glu Gly Leu Phe Thr Val Ala Val Ser Met Met 195 200 205 He Arg Asp Ser Ser He Lys Asn Met Ser Cys Cys He Gin Asn He

210 215 220

Leu Leu Gly Gin Gly Lys Glu Val Glu He Ser Leu Pro Ala Pro Phe 225 230 235 240

Val Pro Arg Leu Thr Pro Trp He Val Ala Val Ala He He Leu Leu 245 250 255

Ala Leu Gly Phe Leu Thr He Gly Ser He Phe Phe Thr Trp Lys Leu 260 265 270

Tyr Lys Glu Arg Ser Ser Leu Arg Lys Lys Glu Phe Gly Ser Lys Glu 275 280 285 Arg Leu Leu Glu Glu Leu Arg Cys Lys Lys Thr Val Leu His Glu Val 290 295 300

Asp Val Thr Leu Asp Pro Asp Thr Ala His Pro His Leu Phe Leu Tyr 305 310 315 320

Glu Asp Ser Lys Ser Val Arg Leu Glu Asp Ser Arg Gin He Leu Pro 325 330 335

Asp Arg Pro Glu Arg Phe Asp Ser Trp Pro Cys Val Leu Gly Arg Glu 340 345 350

Thr Phe Thr Ser Gly Arg His Tyr Trp Glu Val Glu Val Gly Asp Arg 355 360 365 Thr Asp Trp Ala He Gly Val Cys Arg Glu Asn Val Val Lys Lys Gly 370 375 380

Phe Asp Pro Met Thr Pro Asp Asn Gly Phe Trp Ala Val Glu Leu Tyr 385 390 395 400

Gly Asn Gly Tyr Trp Ala Leu Thr Pro Leu Arg Thr Ser Leu Arg Leu 405 410 415

Ala Gly Pro Pro Arg Arg Val Gly Val Phe Leu Asp Tyr Asp Ala Gly 420 425 430

Asp He Ser Phe Tyr Asn Met Ser Asn Gly Ser Leu He Tyr Thr Phe 435 440 445 Pro Ser He Ser Phe Ser Gly Pro Leu Arg Pro Phe Phe Cys Leu Trp 450 455 460

Ser Cys Gly Lys Lys Pro Leu Thr He Cys Ser Thr Ala Asn Gly Pro 465 470 475 480 Glu Lys Val Thr Val He Ala Asn Val Gin Asp Asp He Pro Leu Ser 485 490 495

Pro Leu Gly Glu Gly Cys Thr Ser Gly Asp Lys Asp Thr Leu His Ser 500 505 510 Lys Leu He Pro Phe Ser Pro Ser Gin Ala Ala Pro

515 520

(2) INFORMATION FOR SEQ ID NO: 5:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(n) MOLECULE TYPE, other nucleic acid

(A) DESCRIPTION, /desc = "oligodeoxynucleotide" (ill) HYPOTHETICAL. YES

(iv) ANTI -SENSE: NO

(ix) FEATURE:

(A) NAME/KEY- protem_bmd

(B) LOCATION: 1..10 (D) OTHER INFORMATION: /bound_moιety= "Transcription factor"

(XI ) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATTAGGTAAT 10

Claims

We claim:

1. A purified and isolated DNA fragment comprising a DNA sequence having the biological activity of a butyrophilin promoter.

2. The DNA fragment of claim 1, wherein the DNA sequence comprises at least

one minimal promoter region from the mouse Btn promoter, the bovine BTN promoter, or their

substantial equivalents.

3. The DNA fragment of claim 2, wherein the minimal promoter region is from

the mouse Btn promoter.

4. The DNA fragment of claim 1, wherein the DNA sequence is selected from the

group consisting of:

(a) nucleotides 1 to 4693 of SEQ ID NO:l; and

(b) DNA sequences which are substantial equivalents of the sequences defined in (a).

5. The DNA fragment of claim 4, wherein the DNA sequence further comprises

nucleotides 4694-4922 of SEQ ID NO: l , wherein the nucleotides 4694-4922 are contiguous

with the nucleotides 1 to 4693.

6. The DNA fragment of claim 5, wherein the DNA sequence further comprises

nucleotides 4923-5001 of SEQ ID NO:l, wherein the nucleotides 4923-5001 are contiguous

with the nucleotides 4694-4922.

7. The DNA fragment of claim 6, wherein the DNA sequence further comprises

nucleotides 5002-14180 of SEQ ID NO:l, or its complementary sequence, wherein the

nucleotides 5002-14180 are contiguous with the nucleotides 4923-5001.

8. The DNA fragment of claim 1, wherein the DNA sequence is the bovine BTN

promoter.

9. A rDNA construct for expressing a polypeptide in the mammary gland of a

mammal, the rDNA construct comprising:

(a) a first DNA sequence having the biological activity of the butyrophilin

promoter; and

(b) a second DNA sequence encoding the polypeptide operatively linked to the first

DNA sequence.

10. The rDNA construct of claim 7, wherein the first DNA sequence comprises at

least one minimal butyrophilin promoter region.

11. The rDNA construct of claim 10, wherein the minimal promoter region is from

the mouse Btn promoter, the bovine BTN promoter, or their substantial equivalents.

12. The rDNA construct of claim 9, wherein the first DNA sequence is selected

from the group consisting of: (a) a DNA sequence comprising nucleotides 1 to 4693 of SEQ ID NO: 1 ;

and (b) DNA sequences which are substantial equivalents of the sequence

defined in (a).

13. The rDNA construct of claim 12, wherein the first DNA sequence further

comprises nucleotides 4694-4922 of SEQ ID NO: 1 , or its complementary sequence, contiguous

with the nucleotides 1 to 4693.

14. The rDNA construct of claim 9, further comprising a third DNA sequence encoding a protein signal sequence operatively linked between first and second DNA sequences.

15. The rDNA construct of claim 14, wherein the signal sequence is a milk protein

signal sequence and the third DNA sequence is fused to the second DNA sequence.

16. The rDNA construct of claim 15, wherein the third DNA sequence is selected from the group consisting of:

(a) a DNA sequence comprising nucleotides 4923-5001 of SEQ ID NO: 1 ,

or its complementary sequence, and

(b) DNA sequences which are substantial equivalents of the sequences defined in (a) .

17. A transgenic mammal containing a rDNA construct in at least its mammary epithelial cells, the rDNA construct comprising

(a) a first DNA sequence having the biological activity of a butyrophilin promoter; and

(b) a second DNA sequence encoding a polypeptide operatively linked to the first DNA sequence, the rDNA construct being integrated in such a way that the second DNA sequence is expressed in the mammary gland of the transgenic mammal and the polypeptide is present in the milk of

the mammal.

18. The transgenic mammal of claim 17, wherein the first DNA sequence comprises

at least one minimal butyrophilin promoter region.

19. The transgenic mammal of claim 18, wherein the minimal promoter region is

from the mouse Btn promoter, the bovine BTN promoter, or their substantial equivalents.

20. The transgenic mammal of claim 17, wherein the first DNA sequence is selected

from the group consisting of:

(a) a DNA sequence comprising nucleotides 1 to 4693 of SEQ ID NO: l;

and

(b) DNA sequences which are substantial equivalents of the sequence defined in (a).

21. The transgenic mammal of claim 20, wherein the first DNA sequence further

comprises nucleotides 4694-4922 of SEQ ID NO: l, or its complementary strand, contiguous

with the nucleotides 1 to 4693.

22. The transgenic mammal of claim 17, wherein the rDNA construct further

comprises a third DNA sequence encoding a signal sequence operatively linked between the

first and second DNA sequences.

23. The transgenic mammal of claim 22, wherein the signal sequence is a milk

protein signal sequence and the third DNA sequence is fused to the second DNA sequence.

24. The transgenic mammal of claim 17, wherein the rDNA construct is also present

in the germ cells and all the somatic cells of the transgenic mammal.

25. A method of producing a polypeptide comprising the steps of

(a) producing milk in a transgenic mammal, the mammal containing a rDNA construct in at least its mammary epithelial cells, the rDNA

construct comprising (i) a first DNA sequence having the biological activity of a Btn promoter; and

(ii) a second DNA sequence encoding the polypeptide operatively

linked to the first DNA sequence; the rDNA construct being integrated in such a way that the second DNA sequence is

expressed in the mammary gland of the transgenic mammal and

the polypeptide is present in the milk; and

(b) collecting the milk produced in step (a).

26. The method of claim 25, further comprising:

(c) removing the polypeptide from the collected milk.

27. The method of claim 25, wherein the rDNA construct is also present in the germ

cells and all the somatic cells of the transgenic mammal.

28. A method for detecting a disease state associated with activation of a Btn

promoter in nonlactating mammals comprising detecting expression of butyrophilin mRNA or

protein in a tissue of a nonlactating mammal.

29. The method of claim 28 wherein the disease state is breast cancer and the tissue

is breast cancer.

30. A method for testing the carcinogenicity of a substance comprising comparing

the level of expression of a reporter gene in a recombinant cell in the presence of the substance with the level of expression of the reporter gene in the recombinant cell in the absence of the

substance, the recombinant cell containing a rDNA construct comprising

(a) the first DNA sequence having the biological activity of a butyrophilin

promoter; and (b) a second DNA sequence encoding the reporter gene operatively linked to the first DNA sequence.

31. A purified and isolated DNA fragment comprising a DNA sequence coding for

a polypeptide having the amino sequence of SEQ ID NO:4.

32. A purified and isolated DNA fragment comprising a DNA sequence encoding

mouse butyrophilin, wherein said DNA sequence comprises nucleotides 4694-13199.

33. A purified and isolated DNA fragment comprising a DNA sequence coding for

the promoter and transcriptional unit of the mouse butyrophilin gene, said DNA sequence obtained by a process comprising the steps of:

(a) growing λBtnl (ATCC Deposit No. 97513) on a host bacteria strain to

generate a lysate of λBtn phage particles;

(b) concentrating the λBtnl phage particles;

(c) extracting λBtnl DNA from the concentrated phage particles; and

(d) sequencing the extracted λBtnl DNA.