JP2001505405A - Butyrophilin gene promoter and its use - Google Patents

Abstract

  (57) [Summary] The DNA sequences of the mouse butyrophilin and its promoter are elucidated and analyzed. In addition, the expression of the mouse butyrophilin gene is characterized. In addition, the use of butyrophilin promoters for expression of polypeptides in the milk of transgenic animals and sieving of carcinogenic substances is elucidated.

Description

DETAILED DESCRIPTION OF THE INVENTION Title of invention   Butyrophilin gene promoter and its use Field of the Invention   The present invention relates generally to the butyrophilin gene promoter. More specifically, The present invention relates to the production of non-cognate proteins by the milk of transgenic animals and carcinogens The invention relates to the use of a butyrophilin gene promoter to detect quality. to this Therefore, the applicant filed US Serial No. 60/022, 563 papers The contents are quoted and incorporated. BACKGROUND OF THE INVENTION   Butyrophilin binds the fat-small membrane (FGM) in many types of milk and It is an important essential protein that may play a role in the secretory machinery. Franke et al., J. Cell Biol. 89: 485-494 (1981); Jack and Mather, J. Amer. Biol. Chem. 265: 14481 -14486 (1990); and Jack and Mather, J. Dairy Sci. 76: 3832-3850 (1993); Then, they are quoted and incorporated in their respective sentences. Breast epithelial cells Butyrophilin, which oozes on the surface of the apex, is a glycosylated exopura Smic domains, membrane anchors roughly in the middle of the sequence, and long cells It is a type I glycoprotein containing a tail.   Butyrophilin is derived from the exoplasmic domains B7.1 (CD80) and And B7.2 (CD86) receptor (Linsley et al., Protein Sci. 3: 1341-1343 (1994). )) And the immunoglobulin superfamily (IgSF) (Gar dinier et al. Neurosci. Res. 33, 177-187 (1992)). Those Is characterized by two exoplasmic immunoglobulin-like domains Yes; one of the variable substances (V) or intermediates (I) near the N-terminus (Williams and Barclay, Ann. Rev. Immunol. 6, 381-405 (1988); Harpaz and Ch. othia, J .; Mol. Biol. 238, 528-539 (1994)); Steady state near the membrane anchor It is one of the substance (C) types (Williams and Barclay, 1988). Exoplasmic Other proteins cognate with butyrophilin in the domain are components of myelin sheath (Gardinier et al., 1992) Myelin oligodendrocyte glycoprotein (MOG), and Major Histocompatibility Complex (Miller et al., Proc. Natl. Acad. Sci. U.S.A. 8 8: 4377-4381) and chicken BG antigen (Gardinier et al., 1992; Mill) er et al., 1991). In the IgSF and BG antigen systems Butyrophilin content implies that butyrophilin has an immune effect ing.   The C-terminal cytoplasmic domain of butyrophilin is composed of Zn fingers and coiled It is similar to the C-terminus in the set of proteins contained in the IL domain. this These proteins are associated with nucleic acids or proteins (Bellini et al., J. Cel. l Biol. 131: 563-570 (1995)), ret finger protein (RFP) (Takahas hi et al. Cell Biol. 8: 1853-1856 (1998)), nuclear antigen of Sjogren's syndrome A (SSA / Ro) (Chan et al., J. Clin. Invest. 87: 68-76 (1991)), Africa Frog nuclear element 7 (XNF7) (Reddy et al., Develop. Biol. 148: 107-11). 6 (1991)), PwA33 of Pleurodeles waltl origin (Bellini et al., EMBO J. 12: 1). 07-114 (1993)) and acid finger protein (AFP) (Chu et al., Genomic s 29: 229-239 (1995)). At the DNA level, this cognate region is MOG MHC class I region on chromosome 6 together with the, RFP and butyrophilin genes Region is surrounded by an exon called B30.2 (Vernet et al. , J. et al. Mol. Evol. 37: 600-612 (1993)). Based on the above knowledge, Vernet et al. (19 93) indicates that the butyrophilin gene is compatible with exon I-set in butyrophilin. An ancestral MOG gene generated by the encoding of the Exon with the ancestral RFP gene generated in the B30.2 region of the tyrophilin gene It has been proposed that the mixture has evolved into MHC.   Butyrophilin is clearly present in breast tissue as a maximum during lactation. It is. The expression of the mammary properties of this butyrophilin gene is consistent with the butyrophilin promoter. It is considered to be under the control of Ta. Butyrophilin is associated with milk FGM in many species. Make up important proteins in the total protein bound, eg bovine More than 40% of total FGM binding protein in milk Motor is the breast to produce cognate proteins in transgenic mammalian milk There is an attractiveness as a unique promoter.   Promoters of other breast characteristic genes such as casein, citrate protein, α-la The ctalbumin and β-lactoglobulin genes are found in the milk of transgenic animals. Has been used directly to produce unrelated proteins. Breast characteristics Recent analysis of gene promoters has shown that mammary gland Leading to the same as the key adjustment factors. These elements are binding sites for CTF / NF1 in β-lactoglobulin (Watson et al., Nucl.AcidsRes. 19: 6603-6610 (1991)) and the clear acid protein gene (Li and Rosen, Mol. Cell Biol. 15: 2063-2070 (1995)); Cow α_S2-Oct1 in the casein gene ( Groenen et al., Nuc. Acids Res. 20: 4311-4318 (1992)); β-casei passively Single-stranded nucleic acid binding protein (Altiok and Groner, Mol> Cell B iol. 14: 6004-6012 (1994)); Stimulation of Ets-cognate proteins (Welte et al., Eur . J. Biochem. 223: 997-1006 (1994)) and passively adjusting the disidentified element Element, citrate protein gene (Kolb et al., J. Cellul. Biochem. 56: 245-261 (199 4)), and regulates progesterone-intermediate suppression of the mouse β-casein gene Gestational period specific protein (Lee and Oka, J. Biol. Chem. 267: 5797-5801 (199 2)). Several, including the most thorough study of the rodent β-casein gene promoter The gene is C / EBP (Doppler et al., J. Biol. Chem. 270: 17962-17969 ( 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 the glucocorticoid response element (Raught e t al., 1995). In addition, promoter elements that are fundamentally necessary for cell survival The gene β1,4-galactosyltransferase is derived from AP-2 and CTF / Binding site for regulating the synthesis of NF1 breast 3.9 kb transcript (Rajput et al., J. Biol. Chem. 271: 5131-5142 (1996)).   The basis for breast characterization is a number of poorly understood systems. First, at some sites, the base of the α-lactalbumin gene promoter The sequence called so-called "milk box", where the identification occurs, is the casein gene (L aird et al. Biochem. J. 254: 85-94 (1988)). 1, STAT5 (Meier and Groner, 1994; Raught et al., 1994) and C / E Binding for BP isoforms (Doppler et al., 1995; Raught et al., 1995) Wrap the site. Also, the casein gene associated with blocks A, B and C Also have three protected sequences (Yoshimura, M. and Oka, T.,Gene 78,267-27 Five). Raught et al. (1995) reported that casein gene expression was STAT5 and glucoside. A response consisting of a corticoid response element and various elements including a C / EBP binding site. It has recently been proposed to be controlled by answer elements (CoREs).   First, the inventor concluded that the promoter sequence contains the promoter region and that They have found that they have no significant similarity to the known sequence of the promoter, and Tyrophilin was asexually reproduced and sequenced.   Analysis of the butyrophilin promoter indicates that the butyrophilin promoter is α- and Many latents associated with immune system genes including the β-interferon response element Includes competent regulatory elements, TCF-1 and PU. Sequence is identical to 1 ( PU. 1 is a macrophage, which is a B cell characteristic transducer associated with the ets oncogene. It is a copying factor. See Klemsz et al., Cell 61: 113-125 (1990). ). In addition to the invention The authors report that the region close to the base of the butyrophilin promoter has the same context in T cells. Regulates the expression of mitogen at the macrophage granulocyte colony-stimulating factor site Was found to contain three repetitive elements of GMCSF. Therefore Butiro Filin promoters are also useful for detecting carcinogens. Disclosure of the invention   The present invention relates to the sequence of the 5 'flanking region and the murine butyrophilin gene ( Btn). In particular, the present invention provides a Btn promoter and The present invention provides a transcription control element contained therein.   Accordingly, it is an object of the invention to encode polypeptides having biological activity. Separation and purification of a DNA fragment constituting one DNA sequence.   Another object of the invention is to provide one of the biologically active butyrophilin promoters. Separation and purification of DNA fragments constituting the DNA sequence.   An additional object of the invention is to express a polypeptide in the mammary gland of a mammal. RDNA construction. The rDNA structure has a D symbolizing the desired polypeptide. Contains the butyrophilin promoter linked to affect the NA sequence You. The rDNA structure also affects the DNA sequence encoding the DNA polypeptide. So that the DNA sequence encodes a signal sequence linked in such a manner. Rather , The signal sequence is a milk protein signal sequence. The DNA structure consists of a transcription unit and And / or 3 'flanking sequences in the butyrophilin gene.   A further object of the present invention is to provide a genetically engineered production of the desired polypeptide in the mammary gland Animal offers. This describes the polypeptide in mammalian mammary epithelial cells. Butyrophilin promoter linked to affect the coding DNA sequence This is achieved by incorporating an rDNA structure containing rDNA structure is suckling Subsequent generations, as described above, that are incorporated into the germline of the product Generating the peptide is optional.   Another aspect of the invention relates to the mitogenic properties of substances capable of carcinogenesis for various reasons. Use of mitogens in the butyrophilin promoter to detect gender. For example, substances found in the surrounding environment or separated from food sources are highly carcinogenic. Tested. The mitogenic properties of a substance are related to the butyrophyll in cells exposed to the substance. Detection of butyrophilin mRNA and protein with phosphopromotor activity, or butyrophilin Evaluation by detection of reporter gene production under the control of the filin promoter Be valued.   It is a further object of the invention to feed non-lactating animals for the production of butyrophilin. Diagnosis of disease states such as breast cancer by sieving non-mammalian tissue. BRIEF DESCRIPTION OF THE FIGURES   FIG. 1A shows the sequence of the mouse butyrophilin gene and the 5 'flanking region. Indicates the location of the subclone prepared from λBtn1 used to generate FIG. 1 is a conceptual diagram of a λBtn1 clone.   FIG. 1B shows the murine Btn gene structure showing exon and intron locations. It is a conceptual diagram.   FIG. 1C shows the cDNA subclone used for the sequence of the mouse butyrophilin sCNA. FIG. 3 is a conceptual diagram of a mouse butyrophilin cDNA showing the position of a loan.   2A-C show near the base of the 5 'flanking region of the mouse butyrophilin gene. It shows the position of the prescribed element estimated nearby. BEST MODE FOR CARRYING OUT THE INVENTION   The invention relates to the sequence of the murine butyrophilin gene, and the butyrophilin promoter. The approximate 4.6 kb sequence in the 5 'flanking region, which is also providing. These sequences were obtained from a mouse genomic library described below. Obtained by the clone to be separated.Example 1 Asexual Reproduction of the Mouse Butyrophilin Gene (Stratagene, La Jolla, CA) Sieve with 2.3 kb XhoI-XbaI fragment of the encoded bovine butyrophilin cDNA (Jack and Mather, 1990). Plaque DNA (total of 500,000 pfu) was Transferred to an iron membrane (Dupont, Boston, MA), denatured with 0.5N NaOH, The membrane was prepared using a pre-cleaning solution (Sambrook et al., Molecular Cloning, A Laboratory Manua). l, 2nd Edition, Cold Spring Harbor Laboratory Press, NY, (1989)) For 2 hours at 42 ° C. followed by 42 ° C. in prehybridization solution. After culturing for 6 hours, a random addition method (Feinberg and Vogelstein, Anal. em. 132: 6-13 (1983))⁹cpm / μg specific activity [α-³²P] -dC Hybridization with bovine butyrophilin cDNA fragment labeled with TP And incubated overnight at 42 ° C. in a solution solution. The filter is 2X SSC (Samb rook et al., 1989), then contain 0.1% (w / v) SDS The membrane was washed three times for 20 minutes each with 2 × SSC at 55 ° C. Exposure to X-ray film overnight at 0 ° C. detected. Some potentially positive plaques ,Grand total The asexual germline DNA was detected by sieving 500,000 pfu and was called λBtn1. Was added to the American Type Culture Collection Was deposited as ９97513.   To confirm that λBtn1 contains the mouse butyrophilin gene , Asexually Reproduced DNA (λBtn1), Rat, and Bovine Genomic DNA Samples Is codified by EcoR1 or HindIII and used as a probe for cognate bovine c DNA [³²P] -labeled 2.3 kb XbaI fragment with Southern blot And In each case, digestion with restriction endonucleases [³²P] -labeling A characteristic pattern of DNA fragments hybridized with the probe was obtained. radiation A similar pattern of the substance labeled bands is shown in the genomic DNA and λBtn1 samples. Detected (data not shown). Be able to use the λBtn1 sequence The mouse cDNA probe, mcDNA3, first 396b of exon 7 The encoding of the 3 'end of exon 3 through p (see FIG. 1C) was performed by RT-PCR. Created. Southern similar to those described above that detected this cognate cDNA Blotting confirmed the identity of λBtn1 (data not shown).Example 2: Rat butyrophilin gene sequence   Subcolonies longer than 14 kb of λBtn1 DNA were generated (FIG. 1). A) and arrange for both elements using the fmol sequencing kit from Promega Corp (Madison, WI). Lined up. The radiograph is scanned with a molecular mechanics computing photographic densitometer and distributed. Sunnyvale, CA). Computer program MACAW (S chuler et al., 1991) compiled a maximum length sequence from a sequencing gel. The sequence is shown in SEQ ID NO: 1. All of the Btn sequences are in GenBank Deposited with the database as Association No.U67065. Butyrophilin The promoter contained the first 4,693 nucleotides of SEQ ID NO: 1. In this area The base (first 1750 nucleotides) portion is shown in SEQ ID NO: 2, For example, here the most transcription start (see below) is the renumbering shown at +1 This is illustrated in FIG.Example 3: Format of the mouse butyrophilin gene   Position of the 5 'end of the mouse butyrophilin mRNA. Transcription start site sequence 5'-GGGCTCT Has GTATTTCCCCTAC-3 '(SEQ ID NO: 3)³²Initiator extension using P-labeled initiator Longitudinal analysis and discrimination by 14 day milk secreted mammary epithelial total RNA. This opening Initiator extension analysis applied from Roussel et al. (DNA Cell Biol. 14: 777-788 (1995)) Here, references are cited. The three main labeled products are Btn was obtained by the initiator elongation assay and is shown at position -83 in FIG. Most frequently used sites at nucleotide T, at least -83, -1 8 and +1 (FIG. 2) (SEQ ID NO: residues 4611, 4675, 4694) It starts from the site. All three transcription start sites are normal TATAA and CCAAT Initiation element 5 'to enable initiation of transcription in boxless genes -YYA₊₁NWYY-3 '(Javahery et al., Mol. Cell Biol. 14: 116-127 (1994), reference text) In the vicinity or inside the context. These two of -83 and -19 The sites contain one and three mismatches respectively from the matching sequence, The site is more common A₊₁There are two nucleotides along the start site. Paradoxically The most proximal part at nucleotide +1 although it does not occur most frequently The ranks are perfectly matched internally.   Btn does not have the usual TATAA element, but has two AT-rich sites, positions 5'-TGTAAAT-3 'at nucleotide -49 (nucleotides 4645-4651 of SED ID NO: 1), and And 5'-TCTAAA-3 'at position -106 (nucleotides 4583-4588 of SED ID NO: 1) Is within 20-25 nucleotides, the weaker start site. Other genes As well as other regions coordinate transcription initiation via TATA- and starter binding proteins This is why the start site at position -83 is the most used site. Can be explained. Interestingly, the latter site is MHC class I gene (Le Boutellier, Crit. Rev. Immunol. 14: 89-129 (1994), The sequence 5'-TCTAAA-3 'characterizing the TATA element in the reference 06) is approaching. In addition, many of the milk protein gene promoters And mouse whey protein genes (Campbell and Rosen, Nucl. Acids Res . 12: 8685-8697 (1984)) and the casein gene (Yu-Lee et al., Nucl. Acids R es. 14: 1883-1902 (1986)) containing the sequence 5'-TTTAAAT-3 ' It should be noted that rather has a similar atypical TATAA box is there.   Btn has a TATA sequence (Breathnach and Chambon, Arm. Rev. Biochem. 50: 349-383 (1981), reference) about 50 nucleotides upstream from Lacks the typical CCAAT factor considered in. However, TATA box The sequence 5'-ACAAAGT-3 '(nucleotide of SEQ ID NO: 1) within 50 nucleotides of the base Otide 4597-4603), as well as nucleotides within 30-40 of the end of the TATA box 5′-CCATTT-3 ′ and 5′-CATTT-3 ′ (nucleotides of SEQ ID NO: 1, respectively) Sites with several CCAAT-like abilities (Fig. 2 Double underlined part). According to the sequence of the milk protein gene promoter, Does not have a CCAAT box.   Mapping of the 3 'end of mouse / butyrophilin mRNA by RT-PCR The   The polyadenyl signal sequence in Btn is the stop codon in Btn. Followed by a first latent polyadenyl (polyA) signal sequence (nucleotide of SEQ ID NO: I). To expand the region of the four cDNAs around -Identified using PCR. The expanded product of the expected size is Obtained with the primers 5 'of the A signal sequence. On the other hand, RT-PCR generation An object surrounds the polyA signal sequence or contains nucleotides 13199 (not shown). Was obtained with a primer pair containing the region 3 '. These data are n is the preferred stop signal, 3 'of the transcript End indicates that it is between nucleotides 13,097 and 13,199. You.   The expected boundary between 5 'and 3' of Btn is approximately 8 as an initial gene transcript size. . Evaluated at 40-8.57 kb. 3.50 as the size of the processed mRNA A value of -3.68 kb is evaluated. Subsequent evaluation is based on oligonucleotide testing (data Milk from mouse mammary gland and total RNA 3.7 is the size of rat butyrophilin mRNA determined by northern analysis. kb.   Sequence analysis of the butyrophilin gene sequence revealed that the untranslated region 5 '(5'-UTR) Inversion is simply repeated in the untranslated region 3 '(3'-UTR). Entertainment Deliciously, the 5'-UTR (nucleotides 4807-481 of SEQ ID NO: 1) The repeat sequence in 4) is a 3'-UTR sequence (nucleotide of SEQ ID NO: 1)   12, 556-12, 563). These sequences are Demonstrates an active role in the synthesis, stability or regulation of filin transcription are doing.   Translation of rat butyrophilin mRNA   Predicted rodent butyrophilin amino acid sequence is determined by RT-PCR and rodent Exon / Int from DNA sequence of mouse cDNA generated by gene sequence Ron's boundary was confirmed before extraction. Total RNA is from mouse breast tissue (1 Lactation of the Sun) (Chomczynski and Sacchi, Anal. Biochem. 162: 156-159 (1987)) RT-PCR kit from Perkin Elmer (Perkin Elmer Corp., Branchburg, NJ) according to the procedure described in NJ) for 15 minutes at 42 ° C. Is reverse transcribed into cDNA by cultivation with random hexamers Was. The cDNA, mcDNA 1, 2, 3, 4 (FIG. 1c) was then Generated by enlarging the indicated region of DNA.   The amino acid sequence was determined by the Wisconsin Gene Computer Group (GCG) (SE CDNA sequence verified using the TRANSLATE program of Q ID NO: 4) Predicted. Based on this amino acid sequence, the translation initiation codon AUG is represented by SEQ ID NO: It is predicted to be one nucleotide 4923-4925. This part is a cow Of butyrophilin mRNA is consistent with the predicted position of translation initiation, and Suitable before and after for any eukaryotic gene (Kozak, Nucl. Acids Res. 15: 8125-8248 (1987)) In a relationship. The transcription start site at the extreme end of position 4611 and position 492 3 (SEQ ID NO: 1) and the predicted transcription start site of SEQ ID NO: 1 4 other potential AUG starts at 4650, 4743, 4765, 4776 There are codons. However, the codon at the extreme end of these AUG codons is preferred The other three codons are not in the exact sequence context and the stop codons TAA, TGA, T The AG follows almost immediately in the frame. Most of all In the latter case, the RNA polymerase will start the next potential AUG start site (Koz ak, Nucl. Acids Res. 12: 3873-3893 (1984)).   Comparison of the DNA sequence of Btn with the DNA sequence of butyrophilin cDNA was also performed using IgS Like many other genes in F (Williams and Baclay, 1988), There is a close correlation between the functional units of the protein. Therefore, exo 1 encodes the 5'-UTR and all of the signal sequence. The position of the signal array is shown in the figure. This is indicated by the vertical dashed line in 1B. Exons 2 and 3 are Exon 4 encodes each immunoglobulin-like region of the Code the anchor.   Tissue-specific expression of rat butyrophilin   The previous work suggested that butyrophilin was clearly expressed in breast tissue and expressed Suggest maximum during lactation (described by Mather and Jack, 1993 ). However, this conclusion contaminates the insensitive proteins and RNAs involved. Or the use of immunofluorescence microscopy. Therefore, the mouse MRNA expression in various tissues was determined by a highly sensitive RNA enzyme defense assay. Will be analyzed.   Riboprobe is generated from rat cDNA, mcDNA (Figure 1C), Subcloned into RII (Melton et al., Nucl. Acids Res. 12: 7035-7056 (1984)). Was. For anti-sensory riboprobe, the plasmid was integrated using SP6 RNA. XbaI and RNA were digested for linearization. To sensory revolving probe In addition, the plasmid is constructed using HindIII and RNA integrated using T7 RNA polymerase. And was made linear by cooking. In each case, the RNA is contained in the reaction mixture. [Α-³²P] -dUTP (≧ 800 Ci / mmol). Total RNA Shows 13 tissues (pancreas, intestine, spleen, liver, liver, 3 Balb / c rats) on day 1 of lactation From the kidney, heart, lung, uterus, ovaries, thymus, brain, salivary glands, and breast) (Chomc zynski and sacchi, Anal. Biochem. 162: 156-159 (1987)). Some breast tissue Collected from three Balb / c rats at each stage of development (pregnancy, lactation, regression) Was. Antisensory or sensory riboprobe (2 × 10⁶cpm / sample) is 30 μl 10 μg of total RNA in solution (80% (v / v)) formamide and 1 mM E D TA, 10 mM sodium citrate and 300 mM sodium, pH 6.4 Cultured overnight at 47 ° C. with acetate (Ambion, Austin, Tex.). Within each sample RNA was combined with RNA enzyme 1 (Promega) (5 U / sample) according to the producer's instructions. Cooked at 37 ° C. for 30 minutes. RNA is prepared according to the following standard procedure (Sambrook et al., 198 9) Separation by electrophoresis in 6% (w / v) denatured polyacrylamide gel And recovered. Labeling with RNA from RNA digestion The riboprobe was detected by exposure of the dried gel to X-ray film. .   The size of the antisensory riboprobe depends on the integration of the butyrophilin mRNA into the RNA enzyme. It was expected to protect 625 bp RNA from digestion with nitrogen. Expectation Fragments labeled with radiation of the indicated size were collected from a total of 13 tissues analyzed. Were detected only in breast tissue (not shown). At different stages of development Analysis of breast tissue shows that butyrophilin mRNA is not gland of virgin animals but pregnant, It was shown to be detectable during lactation and regression periods. Butyrophilin mRNA Expression appears to increase significantly in the last half of pregnancy and throughout lactation And remain at a relatively high level.   Analysis of Btn promoter   Btn is specifically expressed in the mammary gland and is associated with MHC or MHC-like genes. (Vernet et al., 1993; Amadou et al., Genomics 26: 9-20 (1995) Investigating similarities between n promoters and regulatory elements of breast traits or immune system genes Inspection was guided. The approximately 1.8 kb Btn5 'flanking sequence shown in SEQ ID NO: 2 is Transcription Factor Data Base (TFD) (Faisst and Meyer, Nucl. Acids Res. 20: 3-26 (1992)), or the whey acid protein, α -Published lactalbumin, β-lactoglobulin, and casein genes Analyzed on the strand by comparison with the sequence.   More than 30 different classes of potential regulatory elements were identified throughout the sequence. other Previously shown to be functional in breast-specific promoters or immune system genes The elements in the assigned Btn are shown in FIG. For clarity, Other elements have been excluded, if not explicitly discussed.   Breast related factors are the general STAT consensus 5'-TTNC (N)_ThreeA It contains three potential STAT binding sites recognized using A-5 '(Ihle and K err, Trends Genet. 11: 69-74 (1995), see citation) (indicated by an asterisk in FIG. 2). Additional STAT binding sites are described by Lamb et al. (Nucl. Acids Res. 23: 3283-328). 9 (1995), refer to the cited example) based on the boundary consensus TT (N)._FiveRecognition using AA (Not shown with an asterisk in FIG. 2). Some C / EBP sites are , Including one between nucleotides 1505 to 1514. One of them Is an incomplete palindrome 5'-ATTAGGTAAT-3 '(SEQ ID NO: : 5). Pregnancy-specific breast nucleus factors (5'-TGAT / ATCA-3 ', Lee and Oka, 19 92, see citations) or single-stranded nucleic acid binding proteins (various For which the sequence is checked, see Altiok and Groner, 1994, citations). There seems to be no site. Btn is a protein related to Ets, NF1 (PU.1 site, Kle Octl (Kemler), a potential binding site for msz et al., 1990, see citations. Others, see EMBO J. 8: 2001-2008 (1989), cited example) and the need for a glycocorticoid response. And a heptamer binding site for the element (1/2 site). There are several YY1 sites and at least 11 GMCSF binding YY1s (Nimer et al., 1990, see citations) (Ye et al., Nucl. Acids Res. 22: 567). 2-5678 (1994), see references). Special feature in whey acid protein gene promoter Two significant negative regulatory elements were identified (Kolb et al., 1994, reference). These points Elements (if you allow one discrepancy each) have the proper context of Btn, Within about 270 nucleotides away from the motor region. Very importantly, The “milk box” area is based on the consensus of Liard et al. (1988) (see citation). Clear CoREs with mixture C / EBP, glucos, not found using the sequence Corticoid response element and STAT5 site (Raught et al., 1995, see citation) Not recognized. Further, FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444-2448 (1988), or BLAST (Altschul et al., J. Mol. Biol. 2). 15: 403-410 (1990)), the promoter of casein, whey acid protein, α-lac Comparison of the talbumin and β-lactoglobulin genes with the 5 ′ flanking region of Btn It showed only limited similarity. The Btn promoter is therefore Genetic It seems to have adjusting elements and new features.   Btn promoter is associated with canonical MHC class I and class II genes Lacking characteristic response elements (Le Boutelier, 1994; Dorn et al., Proc. Natl. Aca d.Sci.U.S.A. 84: 6249-6253 (1987)). However, α- and γ-interfaces And the TCF-1 (see Faisst and Meyer, 1992, cited reference) (see FIG. 2). Consensus sequence for PU. 1 (Klemsz et al., 1990) There are many potential regulatory elements associated with the disease system genes. Inside the base promoter Repetitive elements at three GMCSF sites are recognized and T It has been demonstrated to regulate mitogen-induced expression of GMCSF in cells (Nimer Others, 1990).   Therefore, mouse Btn is desirable in milk of transgenic animals. It can be used to guide protein expression and to select mitogenic mixtures. You. As used herein, the term murine Btn promoter refers to SEQ ID NO: : 1 to 4693 of all sequenced nucleotides, or substantially equivalent Means things. Substantially equivalent is that of SEQ ID NO: 1 under severe conditions D having this sequence integrates into a DNA fragment having nucleotides 1 to 4693 Defined as a DNA sequence that allows for NA fragments.   In addition to regulatory elements found in the promoter region of the gene, tissue-specific expression The regulatory elements present are also located in the transcription unit or 3 'flanking sequence of the gene. It is possible (eg, Charnay et al., Cell 38: 251-263 (1984); Gilles et al. Cel) l 33: 717-728 (1983)). Therefore, the replicated butyrophilin gene is It can be used as a source of such regulatory sequences. For example, if you have a foreign protein The rDNA structure to be expressed was inserted into the first exon of the Btn gene. It may include a DNA sequence encoding for a protein. Preferably, the in What is inserted is normally directed to the secretory pathway involved in butyrophilin secretion into milk. Correctly fused to the Btn signal sequence to target a heterologous protein You.   Example 4 Bovine Butyrophilin Promoter Replication and Analysis   The 5 'untranscribed region of the bovine butyrophilin gene has been described by Jack and Matcher (1990). By standard integration methods using the disclosed bovine butyrophilin cDNA, bovine bovine It can be replicated from the Nom λ library. The cow cattle referred to here as BTN1 Sequence with a mouse promoter and a mouse promoter sequence and a bovine promoter. Comparison of the sequence of the replicating bovine promoter and the regulation contained therein The boundary with the element can be recognized.   Example 5: Creation of synthetic butyrophilin promoter region   All butyrophilin promoters are desirable by those skilled in the art. It will be appreciated that there is no need to provide biological activity. For example, in milk If production of high-quality proteins is desired, the butyrophilin gene in lactating tissue Butyrov needs to respond to transcription factors that regulate the expression of There may be some minimal region or combination of regions within the promoter U. On the other hand, if the purpose is to select mitogenic compounds, Region or in the presence of mitogens, butyrophilin or other genes Butyrophilin promoter that needs to be induced and can be fully induced There will be a combination of regions within. Need to provide the desired biological activity Such a minimal promoter region, which can be provided sufficiently, is It can be recognized by deletion analysis using well-known methods.   Briefly, the deletion construct is a λBt operably linked to a reporter gene. n1 or BTN1 (including, for example, See Example 6). And, in response to various transcription factors, reporter gene expression The quantities are compared in the deleted structure. ΛBtn1 or BTN to provide the desired response One minimal promoter region is either subcloned from the deleted construct or It is made from oligonucleotides synthesized in a moving DNA synthesizer. these The minimal region of the butyrophilin gene, or substantially A DNA sequence derived from the equivalent can be provided. These minimum professionals The motor region is operably linked to the desirably encoded sequence and And can be placed in an expression vector.   Example 6-Structure of butyrophilin: hGH expression vector (Nicholas Institute Diagnostics, San Juan Capistrano, CA) N Can be used to evaluate promoters or promoter regions. Briefly stated Then, a DNA fragment having a butyrophilin promoter or a minimal promoter region Is replicated in the p0GH vector containing the human growth hormone (hGH) structural gene. Lacks eukaryotic promoters. The resulting fusion plasmid is the first breast cell line HGH transfected into the cell and secreted in the middle Using an antibody-based test (Nicholas Institute Diagnostics, # 40-2205) Biologically detected. Since the level of secreted hGH is proportional to the mRNA, Data activity is observable.   In addition to hGH, the encoded chloramphenicol acetyltransferase ( CAT), green fluorescent protein, or other reporter genes such as luminescent enzymes Can be used to evaluate the butyrophilin promoter region. These reporters The detection of gene product products is well known in the art.   Example 7 Transgenic Animal Structure   Different DNA sequences for desired proteins or polypeptides of the early embryo line The generation of transgenic animals with coding is well known in the art. It is performed according to the procedures that have been implemented. For example, Rosen in U.S. Pat. (Transgenic animals) and Clark et al., US Patent 5,322,775 (tiger) Genetic sheep). See the cited examples for all. Generally, the process involves collecting embryos DNA injection into the embryo, alteration of the surviving embryo to substitute for the mother, Has progeny selection for integration and expression. Transgenic animals included in the invention In order to assemble the DNA, the injected DNA must be a butyrophilin promoter or The smallest spot operably linked to a DNA sequence encoding a preferred polypeptide It can be an rDNA construct having a lofilin promoter region. DNA structures are It is also operably linked to a DNA sequence encoding the desired polypeptide. It is desirable to have a signal arrangement.   In addition to constructing early embryo line transgenic animals, the present invention provides Filin promoter or somatic promoter in transgenic mammals Under the control of the protein region, the expression of the desired coding sequence is predicted. Lothar Henningha As described by usen, J. Cell. Biochem., 49: 325-332 (1992), see the cited reference. Animals such as live lactating mammary epithelial cells are injected into a jet injection gun and DNA Generated by rational introduction. Furth, P.A and other differentiated tissues of living animals, See Gene Transfer by Jet Injection into Human Organ Cultures. Mol.Biot echnol 4 (2): 121-127 (Oct. 1995)   Example 8-Disease states associated with butyrophilin expression in non-lactating mammals Detection   As mentioned above, butyrophilin is a member of the IgSF and its cytoplasmic region is Similar to the cytoplasmic region in the zine-finger protein You. Therefore, a block in RNA in non-lactating tissue or in breast tissue of a non-lactating animal. Tyrophilin expression is dependent on cancer or other cancers in which the butyrophilin promoter is active. Can be used to detect disease states.   The principles, preferred embodiments and modes of use of the present invention are described in the foregoing specification. It has been described. However, the invention we are trying to protect here is limited It is considered more descriptive, so it is limited to the form disclosed herein. Not something. To those skilled in the art without departing from the spirit of the invention More embodiments can be varied.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, HU, IL, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, M X, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, ZW (72) Inventor Aug Shelley Elle             United States Maryland 20905,             Silver Spring, Briggs Cha             Ney Road, 905 (72) Inventor Jacques Lucinda JW             ー             United States Maryland 21029,             Clarksville, Hall Shop Law             No. 11912

Claims (1)

[Claims]   1. A refinement comprising a DNA sequence having the biological activity of a butyrophilin promoter DNA fragment isolated.   2. 2. The DNA fragment according to claim 1, wherein the DNA sequence is a mouse Btn plasmid. Motor, bovine BTN promoter, or substantially equivalent It has at least one minimum promoter region.   3. 3. The DNA fragment according to claim 2, wherein the minimum promoter region is a mouse. This is from the Mino Btn promoter.   4. The DNA fragment according to claim 1, wherein the DNA sequence is:   (a) nucleotides 1-4693 of SEQ ID NO: 1,   (b) a DNA sequence substantially equivalent to the sequence defined in (a) above,   Are selected from the group having   5. 5. The DNA fragment according to claim 4, wherein said DNA sequence further comprises SEQ ID NO: Has one nucleotide 4694-4922, and nucleotides 4694-4922 Is adjacent to nucleotides 1-4693.   6. 6. The DNA fragment according to claim 5, wherein said DNA sequence further comprises SEQ ID NO: Having one nucleotide 4923-5001 and nucleotides 4923-5001 Is adjacent to nucleotides 4694-4922.   7. 7. The DNA fragment of claim 6, wherein said DNA sequence further comprises SEQ ID NO: One or more nucleotides 5002-14180 or a supplementary sequence thereof, Tide 5002-14180 is adjacent to nucleotides 4923-5001.   8. 2. The DNA fragment according to claim 1, wherein said DNA sequence is a bovine BTN promoter. Data.   9. In an rDNA structure for expressing a polypeptide in the mammary gland of a mammal, The rDNA structure is   (a) a first DNA sequence having the biological activity of a butyrophilin promoter;   (b) a second encoding a polypeptide operatively linked to said first DNA sequence; 2 DNA sequences,   Is provided.   10. The rDNA structure according to claim 7, wherein the first DNA sequence has at least It has one minimal butyrophilin promoter region.   11. 11. The rDNA structure of claim 10, wherein the minimum promoter region is a mouse. Btn promoter, bovine BTN promoter, or substantially equivalent Because.   12. 10. The rDNA structure of claim 9, wherein the first DNA sequence is   (a) a DNA sequence having nucleotides 1-4693 of SEQ ID NO: 1,   (b) a DNA sequence substantially equivalent to the sequence defined in (a) above,   Are selected from the group having   13. 13. The rDNA structure of claim 12, wherein the first DNA sequence further comprises Nucleotides 4694-4922 of SEQ ID NO: 1, or nucleotides 1-46 It has a supplementary sequence adjacent to 93.   14． 10. The rDNA structure of claim 9, wherein the first and second DNA sequences A third DNA sequence encoding a protein signal sequence that is operatively linked in between. Have.   15. 15. The rDNA structure of claim 14, wherein the signal sequence is a milk protein. Wherein the third DNA sequence is fused to the second DNA sequence. .   16. The rDNA structure of claim 15, wherein the third DNA sequence is   (a) a DNA sequence having nucleotides 4923-5001 of SEQ ID NO: 1, or Is its replenishment sequence,   (b) a DNA sequence substantially equivalent to the sequence defined in (a) above,   Are selected from the group having   17． Trangeni having Γ DNA structure in at least breast epithelial cells In a mammalian mammal, the rDNA structure is   (a) a first DNA sequence having the biological activity of a butyrophilin promoter;   (b) a second encoding a polypeptide operatively linked to said first DNA sequence; 2 DNA sequences, wherein said second DNA sequence is the mammary gland of a transgenic mammal. And the rDNA structure is such that the polypeptide appears in the milk of the mammal. A second DNA integrated in the method;   Having.   18. 18. The transgenic mammal of claim 17, wherein the first DNA The sequence has at least one minimal butyrophilin promoter region.   19. 19. The transgenic mammal of claim 18, wherein said minimal promoter is The promoter region may be mouse Btn, bovine BTN promoter, or substantially the same. And so on.   20. 18. The transgenic mammal of claim 17, wherein the first DNA The array is   (a) a DNA sequence having nucleotides 1-4693 of SEQ ID NO: 1,   (b) a DNA sequence substantially equivalent to the sequence defined in (a) above,   Are selected from the group having   21. 21. The transgenic mammal according to claim 20, wherein the first DNA The sequence may further comprise nucleotides 4694-4922 of SEQ ID NO: 1 or a nucleotide sequence It has a replenishing strand adjacent to Leotide 1-4693.   22. 18. The transgenic mammal of claim 17, wherein said rDNA construct is Further comprising a signal arrangement operatively linked between the first and second DNA sequences. It has a third DNA sequence encoding a sequence.   23. The transgenic mammal of claim 22, wherein the signal sequence is A milk protein signal sequence, wherein the third DNA sequence is fused to the second DNA sequence; Have been.   24. 18. The transgenic mammal of claim 17, wherein said rDNA construct is Construction also occurs in early embryonic cells and in all somatic cells of the transgenic mammal. Appears in   25. In a method for producing a polypeptide,   (a) producing milk in a transgenic mammal, said milk comprising: The mammal has at least a mammary epithelial cell with an rDNA structure, wherein the rDNA structure is ,     (i) a first DNA sequence having a biological activity of a Btn promoter;     (ii) encoding a polypeptide operatively linked to said first DNA sequence       A second DNA sequence, wherein the rDNA structure is a transgenic mammal;       Expressed in the mammary gland of the mammal, wherein the polypeptide has a second DNA sequence that appears in the milk.       Has,   (b) collecting the milk produced in the step (a).   26. The manufacturing method according to claim 25, further comprising:   (c) removing the polypeptide from the collected milk.   27. 26. The method of claim 25, wherein the rDNA structure also comprises an early embryo. It appears in cells and in all somatic cells of transgenic mammals.   28. Butyrophilin mRNA or protein in tissues of non-lactating mammals Diseases associated with Btn promoter activity in non-lactating mammals with expression detection How to detect the condition.   29. 29. The method of claim 28, wherein the disease state is breast cancer and the tissue is breast cancer. Knowledge method.   30. The expression level of the reporter gene in the recombinant cells lacking the substance, Compare reporter gene expression levels in recombinant cells with substance A test method for a carcinogenic substance, wherein the recombinant has a rDNA structure. The cells   (a) a first DNA sequence having the biological activity of a butyrophilin promoter;   (b) encoding the reporter gene operatively linked to the first DNA sequence A second DNA sequence to be converted;   Having.   31. DNA sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4 A purified and isolated DNA fragment having a sequence.   32. Purification and isolation with a DNA sequence encoding rat butyrophilin Wherein the DNA sequence comprises nucleotides 4694-13199. Have.   33. DNA sequence encoding the promoter and the butyrophilin inheritance of the mouse A purified and isolated DNA fragment having a transcription unit of Is obtained in   (a) Variation of host bacteria to produce lysates of λBtn phage particles Growing λBtn1 (ATCC Deposit No.97513) at   (b) collecting λBtn1 phage particles;   (c) extracting λBtn1 DNA from the collected phage particles,   (d) arranging the extracted λBtn1 DNA.