Production of fibrinogen in transgenic animals

Abstract

Materials and methods for producing fibrinogen in transgenic non-human mammals are disclosed. DNA segments encoding A.alpha., B.beta. and .gamma. chains of fibrinogen are introduced into the germ line of a non-human mammal, and the mammal or its female progeny produces milk containing fibrinogen expressed from the introduced DNA segments. Non-human mammalian embryos and transgenic non-human mammals carrying DNA segments encoding heterologous fibrinogen polypeptide chains are also disclosed.

Description

BACKGROUND OF THE INVENTION

The final step in the blood coagulation cascade is the thrombin-catalyzed conversion of the soluble plasma protein fibrinogen to insoluble fibrin. Thrombin cleaves a small peptide (fibrinopeptide A) from one of the three component chains (the A.alpha.-chain) of fibrinogen. Fibrin monomers subsequently polymerize and are cross-linked by activated factor XIII to form a stable clot.

Fibrinogen is a key component of biological tissue glues (see, e.g., U.S. Pat. Nos. 4,377,572 and 4,442,655), which mimic the formation of natural blood clots to promote hemostasis and repair damaged tissue. Tissue glues provide an adjunct or alternative to sutures, staples and other mechanical means for wound closure. However, the principal ingredients of these products (fibrinogen, factor XIII and thrombin) are prepared from pooled human plasma by cryoprecipitation (e.g. U.S. Pat. Nos. 4,377,572; 4,362,567; 4,909,251) or ethanol precipitation (e.g. U.S. Pat. No. 4,442,655) or from single donor plasma (e.g. U.S. Pat. No. 4,627,879; Spotnitz et al., Am. Surg. 55: 166-168, 1989). The resultant fibrinogen/factor XIII preparation is mixed with bovine thrombin immediately before use to convert the fibrinogen to fibrin and activate the factor XIII, thus initiating coagulation of the adhesive.

Commercially available adhesives are of pooled plasma origin. Because blood-derived products have been associated with the transmission of human immunodeficiency virus (HIV), hepatitis virus and other etiologic agents, the acceptance and availability of such adhesives is limited. At present they are not approved for use in the United States.

While the use of autologous plasma reduces the risk of disease transmission, autologous adhesives can only be used in elective surgery when the patient is able to donate the necessary blood in advance.

As noted above, fibrinogen consists of three polypeptide chains, each of which is present in two copies in the assembled molecule. These chains, designated the A.alpha., B.beta. and .gamma.-chains, are coordinately expressed, assembled and secreted by the liver. While it might be expected that recombinant DNA technology could provide an alternative to the isolation of fibrinogen from plasma, this goal has proven to be elusive. The three fibrinogen chains have been individually expressed in E. coli (Lord, DNA 4: 33-38, 1985; Bolyard and Lord, Gene 66: 183-192, 1988; Bolyard and Lord, Blood 73: 1202-1206), but functional fibrinogen has not been produced in a prokaryotic system. Expression of biologically competent fibrinogen in yeast has not been reported. Cultured transfected mammalian cells have been used to express biologically active fibrinogen (Farrell et al., Blood 74: 55a, 1989; Hartwig and Danishefsky, J. Biol. Chem. 266: 6578-6585, 1991; Farrell et al., Biochemistry 30: 9414-9420, 1991), but expression levels have been so low that production of recombinant fibrinogen in commercial quantities is not feasible. Experimental evidence suggests that lower transcription rates in cultured cells as compared to liver may be a factor in the low expression rates achieved to date, but increasing the amount of fibrinogen chain mRNA in transfected BHK cells did not produce corresponding increases in fibrinogen protein secretion (Prunkard and Foster, XIV Congress of the International Society on Thrombosis and Haemostasis, 1993). These latter results suggest that proper assembly and processing of fibrinogen involves tissue-specific mechanisms not present in common laboratory cell lines.

There remains a need in the art for methods of producing large quantities of high quality fibrinogen for use in tissue adhesives and other applications. There is a further need for fibrinogen that is free of blood-borne pathogens. The present invention fulfills these needs and provides other, related advantages.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide commercially useful quantities of recombinant fibrinogen, particularly recombinant human fibrinogen. It is a further object of the invention to provide materials and methods for expressing fibrinogen in the mammary tissue of transgenic animals, particularly livestock animals such as cattle, sheep, pigs and goats.

Within one aspect, the present invention provides a method for producing fibrinogen comprising (a) providing a first DNA segment encoding a secretion signal operably linked to a fibrinogen A.alpha. chain, a second DNA segment encoding a secretion signal operably linked to a fibrinogen B.beta. chain, and a third DNA segment encoding a secretion signal operably linked to a fibrinogen .gamma. chain, wherein each of the first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal; (b) introducing the DNA segments into a fertilized egg of a non-human mammalian species; (c) inserting the egg into an oviduct or uterus of a female of the species to obtain offspring carrying the DNA constructs; (d) breeding the offspring to produce female progeny that express the first, second and third DNA segments and produce milk containing biocompetent fibrinogen encoded by the segments; (e) collecting milk from the female progeny; and (f) recovering the fibrinogen from the milk. Within one embodiment, the egg containing the introduced segments is cultured for a period of time prior to insertion.

Within another aspect, the invention provides a method of producing fibrinogen comprising the steps of (a) incorporating a first DNA segment encoding a secretion signal operably linked to an A.alpha. chain of fibrinogen into a .beta.-lactoglobulin gene to produce a first gene fusion; (b) incorporating a second DNA segment encoding a secretion signal operably linked to a B.beta. chain of fibrinogen into a .beta.-lactoglobulin gene to produce a second gene fusion; (c) incorporating a third DNA segment encoding a secretion signal operably linked to a .gamma. chain of fibrinogen into a .beta.-lactoglobulin gene to produce a third gene fusion; (d) introducing the first, second and third gene fusions into the germ line of a non-human mammal so that the DNA segments are expressed in a mammary gland of the mammal or its female progeny and biocompetent fibrinogen is secreted into milk of the mammal or its female progeny; (e) obtaining milk from the mammal or its female progeny; and (f) recovering the fibrinogen from the milk. Within preferred embodiments, the mammal is a sheep, pig, goat or bovine.

Within another aspect, the invention provides a method for producing fibrinogen comprising the steps of (a) providing a transgenic female non-human mammal carrying in its germline heterologous DNA segments encoding A.alpha., B.beta. and .gamma. chains of fibrinogen, wherein the DNA segments are expressed in a mammary gland of the mammal and fibrinogen encoded by the DNA segments is secreted into milk of the mammal; (b) collecting milk from the mammal; and (c) recovering the fibrinogen from the milk.

Within another aspect, the invention provides a non-human mammalian embryo containing in its nucleus heterologous DNA segments encoding A.alpha., B.beta. and .gamma. chains of fibrinogen. Within a related aspect, the invention provides a transgenic non-human female mammal that produces recoverable amounts of human fibrinogen in its milk.

Within another aspect, the invention provides a method for producing a transgenic offspring of a mammal comprising the steps of (a) providing a first DNA segment encoding a fibrinogen A.alpha. chain, a second DNA segment encoding a fibrinogen B.beta. chain, and a third DNA segment encoding a fibrinogen .gamma. chain, wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in a mammary gland of a host female mammal and secretion into milk of the host female mammal; (b) introducing the DNA segments into a fertilized egg of a mammal of a non-human species; (c) inserting the egg into an oviduct or uterus of a female of the non-human species to obtain an offspring carrying the first, second and third DNA segments. In a related aspect, the invention provides non-human mammals produced according to this process.

Within an additional aspect, the invention provides a non-human mammal carrying its germline DNA segments encoding heterologous A.alpha., B.beta. and .gamma. chains of fibrinogen, wherein female progeny of the mammal express the DNA segments in a mammary gland to produce biocompetent fibrinogen.

These and other aspects of the invention will become evident to the skilled practitioner upon reference to the following detailed description and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the subcloning of a human fibrinogen A.alpha. chain DNA sequence.

FIG. 2 is a partial restriction map of the vector Zem228. Symbols used are MT-1p, mouse metallothionein promoter; SV40t, SV40 terminator; and SV40p, SV40 promoter.

FIG. 3 illustrates the subcloning of a human fibrinogen B.beta. chain DNA sequence.

FIG. 4 illustrates the subcloning of a human fibrinogen .gamma. chain DNA sequence.

FIG. 5 is a partial restriction map of the vector Zem219b. Symbols used are MT-1p, mouse metallothionein promoter; hGHt, human growth hormone terminator; SV40p, SV40 promoter; DHFR, dihydrofolate reductase gene; and SV40t, SV40 terminator.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it will be helpful to define certain terms used herein:

As used herein, the term "biocompetent fibrinogen" is used to denote fibrinogen that polymerizes when treated with thrombin to form insoluble fibrin.

The term "egg" is used to denote an unfertilized ovum, a fertilized ovum prior to fusion of the pronuclei or an early stage embryo (fertilized ovum with fused pronuclei).

A "female mammal that produces milk containing biocompetent fibrinogen" is one that, following pregnancy and delivery, produces, during the lactation period, milk containing recoverable amounts of biocompetent fibrinogen. Those skilled in the art will recognized that such animals will produce milk and therefore the fibrinogen, discontinuously.

The term "progeny" is used in its usual sense to include children and descendants.

The term "heterologous" is used to denote genetic material originating from a different species than that into which it has been introduced, or a protein produced from such genetic material.

Within the present invention, transgenic animal technology is employed to produce fibrinogen within the mammary glands of a host female mammal. Expression in the mammary gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating proteins from other sources. Milk is readily collected, available in large quantities, and well characterized biochemically. Furthermore, the major milk proteins are present in milk at high concentrations (from about 1 to 15 g/l).

From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and are preferred at the proof-of-concept stage), within the present invention it is preferred to use livestock mammals including, but not limited to, pigs, goats, sheep and cattle. Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and the ready availability of equipment for collecting sheep milk. See WO 88/00239 for a comparison of factors influencing the choice of host species. It is generally desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the transgenic line at a later date. In any event, animals of known, good health status should be used.

Fibrinogen produced according to the present invention may be human fibrinogen or fibrinogen of a non-human animal. For medical uses, it is preferred to employ proteins native to the patient. The present invention thus provides fibrinogen for use in both human and veterinary medicine. Cloned DNA molecules encoding the component chains of human fibrinogen are disclosed by Rixon et al. (Biochem. 22: 3237, 1983), Chung et al. (Biochem. 22: 3244, 1983), Chung et al. (Biochem. 22: 3250, 1983), Chung et al. (Adv. Exp. Med. Biol. 281: 39-48, 1990) and Chung et al. (Ann. NY Acad. Sci. 408: 449-456, 1983). Bovine fibrinogen clones are disclosed by Brown et al. (Nuc. Acids Res. 17: 6397, 1989) and Chung et al. (Proc. Natl. Acad. Sci. USA 78: 1466-1470, 1981). Other mammalian fibrinogen clones are disclosed by Murakawa et al. (Thromb. Haemost. 69: 351-360, 1993). Representative sequences of human A.alpha., B.beta. and .gamma. chain genes are shown in SEQ ID NOS: 1, 3 and 5, respectively. Those skilled in the art will recognize that allelic variants of these sequences will exist; that additional variants can be generated by amino acid substitution, deletion, or insertion; and that such variants are useful within the present invention. In general, it is preferred that any engineered variants comprise only a limited number of amino acid substitutions, deletions, or insertions, and that any substitutions are conservative. Thus, it is preferred to produce fibrinogen chain polypeptides that are at least 90%, preferably at least 95, and more preferably 99% or more identical in sequence to the corresponding native chains. The term ".gamma. chain" is meant to include the alternatively spliced .gamma.' chain of fibrinogen (Chung et al., Biochem. 23: 4232-4236, 1984). A human .gamma.' chain amino acid sequence is shown in SEQ ID NO: 6. The shorter .gamma. chain is produced by alternative splicing at nucleotides 9511 and 10054 of SEQ ID NO: 5, resulting in translation terminating after nucleotide 10065 of SEQ ID NO: 5.

To obtain expression in the mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include those genes encoding caseins, beta-lactoglobulin (BLG), .alpha.-lactalbumin, and whey acidic protein. The beta-lactoglobulin promoter is preferred. In the case of the ovine beta-lactoglobulin gene, a region of at least the proximal 406 bp of 5' flanking sequence of the ovine BLG gene (contained within nucleotides 3844 to 4257 of SEQ ID NO:7) will generally be used. Larger portions at the 5' flanking sequence, up to about 5 kbp, are preferred. A larger DNA segment encompassing the 5' flanking promoter region and the legion encoding the 5' non-coding portion of the beta-lactoglobulin gene (contained within nucleotides 1 to 4257 of SEQ ID NO:7) is particularly preferred. See Whitelaw et al., Biochem J. 28: 31-39, 1992. Similar fragments of promoter DNA from other species are also suitable.

Other regions of the beta-lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840, 1988; Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482, 1991; Whitelaw et al., Transgenic Res. 1: 3-13, 1991; WO 89/01343; WO 91/02318). In this regard, it is generally preferred, where possible, to use genomic sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of interest. Within certain embodiments of the invention, the further inclusion of at least some introns from the beta-lactoglobulin gene is preferred. One such region is a DNA segment which provides for intron splicing and RNA polyadenylatiom from the 3' non-coding region of the ovine beta-lactoglobulin gene. When substituted for the natural 3' non-coding sequences of a gene, this ovine beta-lactoglobulin segment can both enhance and stabilize expression levels of the protein or polypeptide of interest. Within other embodiments, the region surrounding the initiation ATG of one or more of the fibrinogen sequences is replaced with corresponding sequences from a milk specific protein gene. Such replacement provides a putative tissue-specific initiation environment to enhance expression. It is convenient to replace the entire fibrinogen chain pre-pro and 5' non-coding sequences with those of, for example, the BLG gene, although smaller regions may be replaced.

For expression of fibrinogen, DNA segments encoding each of the three component polypeptide chains of fibrinogen are operably linked to additional DNA segments required for their expression to produce expression units. Such additional segments include the above-mentioned milk protein gene promoter, as well as sequences which provide for termination of transcription and polyadenylation of mRNA. The expression units will further include a DNA segment encoding a secretion signal operably linked to the segment encoding the fibrinogen polypeptide chain. The secretion signal may be a native fibrinogen secretion signal or may be that of another protein, such as a milk protein. The term "secretion signal" is used herein to denote that portion of a protein that directs it through the secretory pathway of a cell to the outside. Secretion signals are most commonly found at the amino-termini of proteins. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690, 1986; and Meade et al., U.S. Pat. No. 4,873,316, which are incorporated herein by reference.

Construction of expression units is conveniently carried out by inserting a fibrinogen chain sequence into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of a fibrinogen chain (including a secretion signal), thereby creating a gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the expression units in plasmids or other vectors facilitates the amplification of the fibrinogen sequences. Amplification is conveniently carried out in bacterial (e.g. E. coli) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.

In view of the size of the fibrinogen chain genes it is most practical to prepare three separate expression units, mix them, and introduce the mixture into the host. However, those skilled in the art will recognize that other protocols may be followed. For example, expression units for the three chains can be introduced individually into different embryos to be combined later by breeding. In a third approach, the three expression units can be linked in a single suitable vector, such as a yeast artificial chromosome or phage P1 clone. Coding sequences for two or three chains can be combined in polycistronic expression units (see, e.g., Levinson et al., U.S. Pat. No. 4,713,339).

The expression unit(s) is(are) then introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can be accomplished by one of several routes, including microinjection (e.g. U.S. Pat. No 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474, 1988) or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539, 1992). The eggs are then implanted into the oviducts or uteri of pseudopregnant females and allowed to develop to term. Offspring carrying the introduced DNA in their germ line can pass the DNA on to their progeny in the normal, Mendelian fashion, allowing the development of transgenic herds. General procedures for producing transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183, 1988; Wall et al., Biol. Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology: 140-143, 1990; Ebert et al., Bio/Technology: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al. J. Cell. Biochem. 49: 113-120, 1992; and WIPO publications WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-1442, 1985; and Hogan et al. (ibid.). These techniques were subsequently adapted for use with larger animals, including livestock species (see e.g., WIPO publications WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6: 179-183, 1988). To summarize, in the most efficient route used to date in the generation of transgenic mice or livestock, several hundred linear molecules of the DNA of interest are injected into one of the pro-nuclei of a fertilized egg. Injection of DNA into the cytoplasm of a zygote can also be employed.

It is preferred to obtain a balanced expression of each fibrinogen chain to allow for efficient formation of the mature protein. Ideally, the three expression units should be on the same DNA molecule for introduction into eggs. This approach, however, may generate technical problems at, for example, the injection and manipulation stages. For example, the size of fibrinogen expression units may necessitate the use of yeast artificial chromosomes (YACs) or phage P1 to amplify and manipulate the DNA prior to injection. If this approach is followed, segments of DNA to be injected, containing all three expression units, would be very large, thus requiring modification of the injection procedure using, for example, larger bore needles. In a more simple approach, a mixture of each individual expression unit is used. It is preferred to combine equimolar amounts of the three expression units, although those skilled in the art will recognize that this ratio may be varied to compensate for the characteristics of a given expression unit. Some expression, generally a reduced level, will be obtained when lesser molar amounts of one or two chains are used, and expression efficiencies can generally be expected to decline in approximate proportion to the divergence from the preferred equimolar ratio. In any event, it is preferred to use a mixture having a ratio of A.alpha.:B.beta.:.gamma. expression units in the range of 0.5-1:0.5-1:0.5-1. When the ratio is varied from equimolar, it is preferred to employ relatively more of the B.beta. expression unit. Alternatively, one or a mixture of two of the expression units is introduced into individual eggs. However, animals derived by this approach will express only one or two fibrinogen chains. To generate an intact fibrinogen molecule by this approach requires a subsequent breeding program designed to combine all three expression units in individuals of a group of animals.

In general, female animals are superovulated by treatment with follicle stimulating hormone, then mated. Fertilized eggs are collected, and the heterologous DNA is injected into the eggs using known methods. See, for example, U.S. Pat No. 4,873,191; Gordon et al, Proc. Natl. Acad. Sci. USA 77: 7380-7384, 1980; Gordon and Ruddle, Science 214: 1244-1246, 1981; Palmiter and Brinster, Cell. 41: 343-345, 1985; Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985; Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al. Bio/Technology 6:179-183, 1988; Wall et al., Biol. Reprod. 32: 645-651, 1985; Buhler et al., Bio/Technology 8: 140-143, 1990; Ebert et al., Bio/Technology 9: 835-838, 1991; Krimpenfort et al., Bio/Technology 9: 844-847, 1991; Wall et al., J. Cell. Biochem. 49: 113-120, 1992; WIPO publications WO 88/00239, WO 90/05118, and WO 92/11757; and GB 87/00458, which are incorporated herein by reference.

For injection into fertilized eggs, the expression units are removed from their respective vectors by digestion with appropriate restriction enzymes. For convenience, it is preferred to design the vectors to that the expression units are removed by cleavage with enzymes that do not cut either within the expression units or elsewhere in the vectors. The expression units are recovered by conventional methods, such as electro-elution followed by phenol extraction and ethanol precipitation, sucrose density gradient centrifugation, or combinations of these approaches.

DNA is injected into eggs essentially as described in Hogan et al., ibid. In a typical injection, eggs in a dish of an embryo culture medium are located using a stereo room microscope (.times.50 or .times.63 magnification preferred). Suitable media include Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid) or bicarbonate buffered media such as M2 or M16 (available from Sigma Chemical Co., St Louis, USA) or synthetic oviduct medium (disclosed below). The eggs are secured and transferred to the center of a glass slide on an injection rig using, for example, a drummond pipette complete with capillary tube. Viewing at lower (e.g. .times.4) magnification is used at this stage. Using the holding pipette of the injection rig, the eggs are positioned centrally on the slide. Individual eggs are sequentially secured to the holding pipette for injection. For each injection process, the holding pipette/egg is positioned in the center of the viewing field. The injection needle is then positioned directly below the egg. Preferably using .times.40 Nomarski objectives, both manipulator heights are adjusted to focus both the egg and the needle. The pronuclei are located by rotating the egg and adjusting the holding pipette assembly as necessary. Once the pronucleus has been located, the height of the manipulator is altered to focus the pronuclear membrane. The injection needle is positioned below the egg such that the needle tip is in a position below the center of the pronucleus. The position of the needle is then altered using the injection manipulator assembly to bring the needle and the pronucleus into the same focal plane. The needle is moved, via the joy stick on the injection manipulator assembly, to a position to the right of the egg. With a short, continuous jabbing movement, the pronuclear membrane is pierced to leave the needle tip inside the pronucleus. Pressure is applied to the injection needle via the glass syringe until the pronucleus swells to approximately twice its volume. At this point, the needle is slowly removed. Reverting to lower (e.g. .times.4) magnification, the injected egg is moved to a different area of the slide, and the process is repeated with another egg.

After the DNA is injected, the eggs may be cultured to allow the pronuclei to fuse, producing one-cell or later stage embryos. In general, the eggs are cultured at approximately the body temperature of the species used in a buffered medium containing balanced salts and serum. Surviving embryos are then transferred to pseudopregnant recipient females, typically by inserting them into the oviduct or uterus, and allowed to develop to term. During embryogenesis, the injected DNA integrates in a random fashion in the genomes of a small number of the developing embryos.

Potential transgenic offspring are screened via blood samples and/or tissue biopsies. DNA is prepared from these samples and examined for the presence of the injected construct by techniques such as polymerase chain reaction (PCR; see Mullis, U.S. Pat. No. 4,683,202) and Southern blotting (Southern, J. Mol. Biol. 98:503, 1975; Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982). Founder transgenic animals, or G0s, may be wholly transgenic, having transgenes in all of their cells, or mosaic, having transgenes in only a subset of cells (see, for example, Wilkie et al., Develop. Biol. 118: 9-18, 1986). In the latter case, groups of germ cells may be wholly or partially transgenic. In the latter case, the number of transgenic progeny from a founder animal will be less than the expected 50% predicted from Mendelian principles. Founder G0 animals are grown to sexual maturity and mated to obtain offspring, or G1s. The G1s are also examined for the presence of the transgene to demonstrate transmission from founder G0 animals. In the case of male G0s, these may be mated with several non-transgenic females to generate many offspring. This increases the chances of observing transgene transmission. Female G0 founders may be mated naturally, artificially inseminated or superovulated to obtain many eggs which are transferred to surrogate mothers. The latter course gives the best chance of observing transmission in animals having a limited number of young. The above-described breeding procedures are used to obtain animals that can pass the DNA on to subsequent generations of offspring in the normal, Mendelian fashion, allowing the development of, for example, colonies (mice), flocks (sheep), or herds (pigs, goats and cattle) of transgenic animals.

The milk from lactating G0 and G1 females is examined for the expression of the heterologous protein using immunological techniques such as ELISA (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and Western blotting (Towbin et al., Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979). For a variety of reasons known in the art, expression levels of the heterologous protein will be expected to differ between individuals.

A satisfactory family of animals should satisfy three criteria: they should be derived from the same founder G0 animal; they should exhibit stable transmission of the transgene; and they should exhibit stable expression levels from generation to generation and from lactation to lactation of individual animals. These principles have been demonstrated and discussed (Carver et al., Bio/Technology 11: 1263-1270, 1993). Animals from such a suitable family are referred to as a "line." Initially, male animals, G0 or G1, are used to derive a flock or herd of producer animals by natural or artificial insemination. In this way, many female animals containing the same transgene integration event can be quickly generated from which a supply of milk can be obtained.

The fibrinogen is recovered from milk using standard practices such as skimming, precipitation, filtration and protein chromatography techniques.

Fibrinogen produced according to the present invention is useful within human and veterinary medicine, such as in the formulation of surgical adhesives. Adhesives of this type are known in the art. See, for example, U.S. Pat. Nos. 4,377,572; 4,442,655; 4,462,567; and 4,627,879, which are incorporated herein by reference. In general, fibrinogen and factor XIII are combined to form a first component that is mixed just prior to use with a second component containing thrombin. The thrombin converts the fibrinogen to fibrin, causing the mixture to gel, and activates the factor XIII. The activated factor XIII cross links the fibrin to strengthen and stabilize the adhesive matrix. Such adhesives typically contain from about 30 mg/ml to about 100 mg/ml fibrinogen and from about 50 .mu.g/ml to about 500 .mu.g/ml factor XIII. They may also contain additional ingredients, such as aprotinin, albumin, fibronectin, bulking agents, and solubilizers. Methods for producing factor XIII are known in the art. See, for example, U.S. Pat No. 5,204,447. The fibrinogen is also useful for coating surfaces of polymeric articles, e.g. synthetic vascular grafts, as disclosed in U.S. Pat. No. 5,272,074 (incorporated herein by reference).

The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example I

The multiple cloning site of the vector pUC18 (Yanisch-Perron et al., Gene 33:103-119, 1985) was removed and replaced with a synthetic double stranded oligonucleotide (the strands of which are shown in SEQ ID NO: 8 and SEQ ID NO: 27) containing the restriction sites Pvu I/Mlu I/Eco RV/Xba I/Pvu I/Mlu I, and flanked by 5' overhangs compatible with the restriction sites Eco RI and Hind III. pUC18 was cleaved with both Eco RI and Hind III, the 5' terminal phosphate groups were removed with calf intestinal phosphatase, and the oligonucleotide was ligated into the vector backbone. The DNA sequence across the junction was confirmed by sequencing, and the new plasmid was called pUCPM.

The .beta.-lactoglobulin (BLG) gene sequences from pSS1tgXS (disclosed in WIPO publication WO 88/00239) were excised as a Sal I-Xba I fragment and recloned into the vector pUCPM that had been cut with Sal I and Xba I to construct vector pUCXS. pUCXS is thus a pUC18 derivative containing the entire BLG gene from the Sal I site to the Xba I site of phage SS1 (Ali and Clark, J. Mol. Biol. 199: 415-426, 1988).

The plasmid pSS1tgSE (disclosed in WIPO publication WO 88/00239) contains a 1290 bp BLG fragment flanked by Sph I and EcoR I restriction sites, a region spanning a unique Not I site and a single Pvu II site which lies in the 5' untranslated leader of the BLG mRNA. Into this Pvu II site was ligated a double stranded, 8 bp DNA linker (5'-GGATATCC-3') encoding the recognition site for the enzyme Eco RV. This plasmid was called pSS1tgSE/RV. DNA sequences bounded by Sph I and Not I restriction sites in pSS1tgSE/RV were excised by enzymatic digestion and used to replace the equivalent fragment in pUCXS. The resulting plasmid was called pUCXSRV. The sequence of the BLG insert in pUCSXRV is shown in SEQ ID NO: 7, with the unique Eco RV site at nucleotide 4245 in the 5' untranslated leader region of the BLG gene. This site allows insertion of any additional DNA sequences under the control of the BLG promoter 3' to the transcription initiation site.

Using the primers BLGAMP3 (5'-TGG ATC CCC TGC CGG TGC CTC TGG-3'; SEQ ID NO: 9) and BLGAMP4 (5'-AAC GCG TCA TCC TCT GTG AGC CAG-3'; SEQ ID NO: 10) a PCR fragment of approximately 650 bp was produced from sequences immediately 3' to the stop codon of the BLG gene in pUCXSRV. The PCR fragment was engineered to have a BamH I site at its 5' end and an Mlu I site at its 3' end and was cloned as such into BamH I and Mlu I cut pGEM7zf(+) (Promega) to give pDAM200(+).

pUCXSRV was digested with Kpn I, and the largest, vector containing band was gel purified. This band contained the entire pUC plasmid sequences and some 3' non-coding sequences from the BLG gene. Into this backbone was ligated the small Kpn I fragment from pDAM200(+) which, in the correct orientation, effectively engineered a BamH I site at the extreme 5' end of the 2.6 Kbp of the BLG 3' flanking region. This plasmid was called pBLAC200. A 2.6 Kbp Cla I-Xba I fragment from pBLAC200 was ligated into Cla I-Xba I cut pSP72 vector (Promega), thus placing an EcoR V site immediately upstream of the BLG sequences. This plasmid was called pBLAC210.

The 2.6 Kbp Eco RV-Xba I fragment from pBLAC210 was ligated into Eco RV-Xba I cut pUCXSRV to form pMAD6. This, in effect, excised all coding and intron sequences from pUCXSRV, forming a BLG minigene consisting of 4.3 Kbp of 5' promoter and 2.6 Kbp of 3' downstream sequences flanking a unique EcoR V site. An oligonucleotide linker (ZC6839: ACTACGTAGT; SEQ ID NO: 11) was inserted into the Eco RV site of pMAD6. This modification destroyed the Eco RV site and created a Sna BI site to be used for cloning purposes. The vector was designated pMAD6-Sna. Messenger RNA initiates upstream of the Sna BI site and terminates downstream of the Sna BI site. The precursor transcript will encode a single BLG-derived intron, intron 6, which is entirely within the 3' untranslated region of the gene.

Example II

Clones encoding the individual fibrinogen chains were obtained from the laboratory of Dr. Earl W. Davie, University of Washington, Seattle. A genomic fibrinogen A.alpha.-chain clone (Chung et al., 1990, ibid.) was obtained from the plasmid BS4. This plasmid contains the A.alpha. clone inserted into the Sal I and Bam HI sites of the vector pUC18, but lacks the coding sequence for the first four amino acids of the A.alpha. chain. A genomic B.beta.-chain DNA (Chung et al., ibid.) was isolated from a lambda Charon 4A phage clone (designated .beta..lamda.4) as two EcoRI fragments of ca. 5.6 Kbp each. The two fragments were cloned separately into pUC19 that had been digested with Eco RI and treated with calf intestinal phosphatase. The resulting clones were screened by digestion with the restriction enzyme Pvu II to distinguish plasmids with the 5' and 3' Bp inserts (designated Beta5'RI/puc and Beta3'RI/puc, respectively). Genomic .gamma.-chain clones were isolated as described by Rixon et al. (Biochemistry 24: 2077-2086, 1985). Clone p.gamma.12A9 comprises 5' non-coding sequences and approximately 4535 bp of .gamma.-chain coding sequence. Clone p.gamma.12F3 comprises the remaining coding sequence and 3' non-coding nucleotides. Both are pBR322-based plasmids with the fibrinogen sequences inserted at the EcoRI site. These plasmids were used as templates for the respective PCR reactions.

The fibrinogen chain coding sequences were tailored for insertion into expression vectors using the polymerase chain reaction (PCR) as generally described by Mullis (U.S. Pat. No. 4,683,202). This procedure removed native 5' and 3' untranslated sequences, added a 9 base sequence (CCT GCA GCC) upstream of the first ATG of each coding sequence, supplied the first four codons for the A.alpha.-chain sequence, removed an internal Mlu I site in the A.alpha. sequence and added restriction sites to facilitate subsequent cloning steps.

Referring to FIG. 1, the 5' end of the A.alpha. coding sequence was tailored in a PCR reaction containing 20 pmole for each of primers ZC6632 (SEQ ID NO: 12) and ZC6627 (SEQ ID NO: 13), approximately 10 ng of plasmid BS4 template DNA, 10 .mu.l of a mix containing 2.5 mM each dNTP, 7.5 .mu.l 10.times. Pyrococcus furiosus (Pfu) DNA polymerase buffer #1 (200 mM Tris-HCl, pH 8.2, 100 mM KCl, 60 mM (NH.sub.4).sub.2SO.sub.4, 20 mM MgCl.sub.2, 1% Triton X-100, 100 .mu.g/ml nuclease free bovine serum albumin)(Stratagene, La Jolla, Calif.), and water to 75 .mu.l. The mixture was heated to 94.degree. C. in a DNA thermal cycler (Perkin-Elmer Corp., Norwalk, Conn.). To the heated mixture was added 25 .mu.l of a mixture containing 2.5 .mu.l 10.times.Pfu buffer #1, 22 .mu.l H.sub.2O and 1 .mu.l 2.5 units/.mu.l Pfu DNA polymerase (Stratagem). The reactions were run in a DNA thermal cycler (Perkin-Elmer) for five cycles of 94.degree., 45 seconds; 40.degree., 90 seconds; 72.degree., 120 seconds; 20 cycles of 94.degree., 45 seconds; 45.degree., 90 seconds; 72.degree., 120 seconds; then incubated at 72.degree. for 7 minutes. The 5' PCR-generated fragment was digested with Bam HI and Hind III, and the Bam HI-Hind III fragment was then ligated to an internal 2.91 Kbp Hind III-Xba I fragment and Bam HI, Xba I-digested pUC18. PCR-generated exon sequences were sequenced.

Referring again to FIG. 1, the 3' end of the A.alpha. coding sequence was tailored in a series of steps in which the Mlu I site 563 bases upstream from the stop codon of the A.alpha. sequence was mutated using an overlap extension PCR reaction (Ho et al., Gene 77: 51-59, 1989). In the first reaction 40 pmole of each of primers ZC6521 (SEQ ID NO: 14) and ZC6520 (SEQ ID NO: 15) were combined with approximately 10 ng of plasmid BS4 template DNA in a reaction mixture as described above. The reaction was run for 5 cycles of 94.degree., 45 seconds; 40.degree., 60 seconds; 72.degree., 120 seconds; 15 cycles of 94.degree., 45 seconds; 45.degree., 60 seconds; 72.degree., 120 seconds; then incubated at 72.degree. for 7 minutes. A second reaction was carried out in the same manner using 40 pmole of each of primers ZC6519 (SEQ ID NO: 16) and ZC6518 (SEQ ID NO: 17) and BS4 as template. The PCR-generated DNA fragments from the first and second reactions were isolated by gel electrophoresis and elution from the gel. Approximately 1/10 of each recovered reaction product was combined with 40 pmole of each of primers ZC6521 (SEQ ID NO: 14) and ZC6518 (SEQ ID NO: 17) in a PCR reaction in which the complementary 3' ends of each fragment (containing the single base change) annealed and served as a primer for the 3' extension of the complementary strand. PCR was carried out using the same reaction conditions as in the first and second 3' PCR steps. The reaction product was then digested with Xba I and Bam HI, and the Xba I-Bam HI fragment was cloned into Xba I, Bam HI-digested pUC18. PCR-generated exons were sequenced.

As shown in FIG. 1, the 5' Bam HI-Xba I fragment (3.9 Kbp) and the 3' Xba I-Bam HI fragment (1.3 Kbp) were inserted into the Bam HI site of the vector Zem228. Zem228 is a pUC18 derivative comprising a Bam HI cloning site between a mouse MT-1 promoter and SV40 terminator, and a neomycin resistance marker flanked by SV40 promoter and terminator sequences. See European Patent Office Publication EP 319,944 and FIG. 2. The entire A.alpha. coding sequence was isolated from the Zem228 vector as an Sna BI fragment, which was inserted into the Sna BI site of the plasmid pMAD6-Sna.

Referring to FIG. 3, the 5' end of the B.beta.-chain was tailored by PCR using the oligonucleotides ZC6629 (SEQ ID NO: 18), ZC6630 (SEQ ID NO: 19) and ZC6625 (SEQ ID NO: 20). These primers were used in pairwise combinations (ZC6629+ZC6625 or ZC6630+ZC6625) to generate B.beta. coding sequences beginning at the first ATG codon (position 470 in SEQ ID NO: 3)(designated N1-Beta) or the third ATG codon (position 512 in SEQ ID NO: 3)(designated N3-Beta). Approximately 5 ng of Beta5'RI/puc template DNA was combined with 20 pmole of each of the primers (N1-Beta:ZC6629, SEQ ID NO: 18+ZC6625, SEQ ID NO: 20; or N3-Beta:ZC6630, SEQ ID NO: 19+ZC6625, SEQ ID NO: 20) in a reaction mixture as described above. The mixtures were incubated for 5 cycles of 94.degree., 45 seconds; 40.degree., 120 seconds; (N1-Beta) or 90 seconds (N3-Beta); 72.degree., 120 seconds; 20 cycles of 94.degree., 45 seconds; 45.degree., 120 seconds; (N1-Beta) or 90 seconds (N3-Beta); 72.degree., 120 seconds; then incubated at 72.degree. for 7 minutes. The two reaction products N1, 555 bp or N3, 510 bp) were each digested with Eco RI and Bgl II, and the fragments were ligated to the internal Bgl II-Xba I fragment and Eco RI+Xba I-digested pUC19. The 3' end of the B.beta. sequence was tailored in a reaction mixture as described above using the oligonucleotide primers ZC6626 (SEQ ID NO: 21) and ZC6624 (SEQ ID NO: 22) and approximately 5 ng of Beta3'RI/puc template. The mixtures were incubated for 5 cycles of 94.degree., 45 seconds; 40.degree., 90 seconds; 72.degree., 120 seconds; 15 cycles of 94.degree., 45 seconds; 45.degree., 90 seconds; 72.degree., 120 seconds; then incubated at 72.degree. for 7 minutes. A 990 bp Bgl II-Eco RI fragment was isolated. This 3' fragment was ligated to the adjacent coding fragment (340 bp, SphI-Bgl II) and Sph I+Eco RI-digested pUC19. The 3' and 5' PCR-generated exons were sequenced. A third intermediate vector was constructed by combining two internal fragments (4285 bp Xba I-Eco RI and 383 kb Eco RI-Sph I) in Xba I+Sph I-digested pUC19. The entire B.beta. coding sequence (two forms) was then assembled by ligating one of the 5' Eco RI-Xba I fragments, the internal Xba I-Sph I fragment, the 3' Sph I-Eco RI fragment and Eco RI-digested vector pUC19. The B.beta. sequence was then isolated as a 7.6 Kbp Sna BI fragment and inserted into the Sna BI site of pMAD6-Sna.

Referring to FIG. 4, the 5' end of the gamma chain sequence was tailored by PCR using the oligonucleotide primers ZC6514 (SEQ ID NO: 23) and ZC6517 (SEQ ID NO: 24) and approximately 50 ng of p.gamma.12A9 as template. The PCR reaction was run as described above using 40 pM of each primer. The reaction was run for 5 cycles of 94.degree., 45 seconds; 40.degree., 60 seconds, 72.degree., 120 seconds, followed by 15 cycles of 94.degree., 45 seconds; 45.degree., 60 seconds; 72.degree., 120 seconds. The resulting 213 by fragment was digested with Bam HI and Spe I, and the resulting restriction fragment was ligated with the adjacent downstream 4.4 kb Spe I-Eco RI fragment and Bam HI+Eco RI digested pUC19. The 3' end of the gamma chain sequence was tailored using oligonucleotide primers ZC6516 (SEQ ID NO: 25) and ZC6515 (SEQ ID NO: 26) using 40 pM of each primer, approximately 50 ng of p.gamma.12F3 template and the same thermal cycling schedule as used for the 5' fragment. The resulting 500 bp fragment was digested with Spe I and Bam HI, and the resulting restriction fragment was ligated with the upstream 2.77 kb Eco RI-Spe I fragment and Eco RI+Bam HI-digested pUC19. All PCR-generated exons were sequenced. The entire .gamma.'-chain coding sequence was then assembled by ligating a 4.5 Kbp Bam HI-Eco RI 5' fragment, a 1.1 Kbp Eco RI-Pst I internal fragment and a 2.14 Kbp Pst I-Xba I 3' fragment in Bam HI+Xba I-digested Zem219b. Zem219b is a pUC18-derived vector containing a mouse metallothionein promoter and a DHFR selectable marker operably linked to an SV40 promoter (FIG. 5). Plasmid Zem219b has been deposited with American Type Culture Collection as an E. coli XL1-blue transformant under Accession No. 68979. The entire .gamma.-chain coding sequence was then isolated as a 7.8 Kbp Sna B1 fragment and inserted into the Sna BI site of pMAD6-Sna.

Example III

Mice for initial breeding stocks (C57BL6J, CBACA) were obtained from Harlan Olac Ltd. (Bicester, UK). These were mated in pairs to produce F1 hybrid cross (B6CBAF1) for recipient female, superovulated females, stud males and vasectomized males. All animals were kept on a 14 hour light/10 hour dark cycle and fed water and food (Special Diet Services RM3, Edinburgh, Scotland) ad libitum.

Transgenic mice were generated essentially as described in Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986, which is incorporated herein by reference in its entirety. Female B6CBAF1 animals were superovulated at 4-5 weeks of age by an i.p. injection of pregnant mares' serum gonadotrophin (FOLLIGON, Vet-Drug, Falkirk, Scotland) (5 iu) followed by an i.p. injection of human chorionic gonadotrophin (CHORULON, Vet-Drug, Falkirk, Scotland) (5 iu) 45 hours later. They were then mated with a stud male overnight. Such females were next examined for copulation plugs. Those that had mated were sacrificed, and their eggs were collected for microinjection.

DNA was injected into the fertilized eggs as described in Hogan et al. (ibid.) Briefly, each of the vectors containing the A.alpha., B.beta. and .gamma. expression units was digested with Mlu I, and the expression units were isolated by sucrose gradient centrifugation. All chemicals used were reagent grade (Sigma Chemical Co., St. Louis, Mo., U.S.A.), and all solutions were sterile and nuclease-free. Solutions of 20% and 40% sucrose in 1M NaCl, 20 mM Tris pH 8.0, 5 mM EDTA were prepared using UHP water and filter sterilized. A 30% sucrose solution was prepared by mixing equal volumes of the 20% and 40% solutions. A gradient was prepared by layering 0.5 ml steps of the 40%, 30% and 20% sucrose solutions into a 2 ml polyallomer tube and allowed to stand for one hour. 100 .mu.l of DNA solution (max. 8 .mu.g DNA) was loaded onto the top of the gradient, and the gradient was centrifuged for 17-20 hours at 26,000 rpm, 15.degree. C. in a Beckman TL100 ultracentrifuge using a TLS-55 rotor (Beckman Instruments, Fullerton, Calif., USA). Gradients were fractionated by puncturing the tube bottom with a 20 ga. needle and collecting drops in a 96 well microliter plate. 3 .mu.l aliquots were analyzed on a 1% agarose mini-gel. Fractions containing the desired DNA fragment were pooled and ethanol precipitated overnight at -20.degree. C. in 0.3M sodium acetate. DNA pellets were resuspended in 50-100 .mu.l UHP water and quantitated by fluorimetry. The expression units were diluted in Dulbecco's phosphate buffered saline without calcium and magnesium (containing, per liter, 0.2 g KCl, 0.2 g KH.sub.2PO.sub.4, 8.0 g NaCl, 1.15 g Na.sub.2HPO.sub.4), mixed (using either the N1-Beta or N3-Beta expression unit) in a 1:1:1 molar ratio, concentration adjusted to about 6 .mu.g/ml, and injected into the eggs (.about.2 pl total DNA solution per egg).

Recipient females of 6-8 weeks of age are prepared by mating B6CBAF1 females in natural estrus with vasectomized males. Females possessing copulation plugs are then kept for transfer of microinjected eggs.

Following birth of potential transgenic animals, tail biopsies are taken, under anesthesia, at four weeks of age. Tissue samples are placed in 2 ml of tail buffer (0.3M Na acetate, 50 mM HCl, 1.5 mM MgCl.sub.2, 10 mM Tris-HCl, pH 8.5, 0.5% NP40, 0.5% Tween 20) containing 200 .mu.g/ml proteinase K (Boehringer Mannheim, Mannheim, Germany) and vortexed. The samples are shaken (250 rpm) at 55.degree.-60.degree. for 3 hours to overnight. DNA prepared from biopsy samples is examined for the presence of the injected constructs by PCR and Southern blotting. The digested tissue is vigorously vortexed, and 5 .mu.l aliquots are placed in 0.5 ml microcentrifuge tubes. Positive and negative tail samples are included as controls. Forty .mu.l of silicone oil (BDH, Poole, UK) is added to each tube, and the tubes are briefly centrifuged. The tubes are incubated in the heating block of a thermal cycler (e.g. Omni-gene, Hybaid, Teddington, UK) to 95.degree. C. for 10 minutes. Following this, each tube has a 45 .mu.l aliquot of PCR mix added such that the final composition of each reaction mix is: 50 mM KCl; 2 mM MgCl.sub.2; 10 mM Tris-HCl (pH 8.3); 0.01% gelatin; 0.1% NP40, 10% DMSO; 500 nM each primer, 200 .mu.M dNTPs; 0.02 U/.mu.l Taq polymerase (Boehringer Mannheim, Mannheim, Germany). The tubes are then cycled through 30 repeated temperature changes as required by the particular primers used. The primers may be varied but in all cases must target the BLG promoter region. This is specific for the injected DNA fragments because the mouse does not have a BLG gene. Twelve .mu.l of 5.times. loading buffer containing Orange G marker dye (0.25% Orange G [Sigma] 15% Ficoll type 400 [Pharmacia Biosystems Ltd., Milton Keynes, UK]) is then added to each tube, and the reaction mixtures are electrophoresed on a 1.6% agarose gel containing ethidium bromide (Sigma) until the marker dye has migrated 2/3 of the length of the gel. The gel is visualized with a UV light source emitting a wavelength of 254 nm. Transgenic mice having one or more of the injected DNA fragments are identified by this approach.

Positive tail samples are processed to obtain pure DNA. The DNA samples are screened by Southern blotting using a BLG promoter probe (nucleotides 2523-4253 of SEQ ID NO: 7). Specific cleavages with appropriate restriction enzymes (e.g. Eco RI) allow the distinction of the three constructs containing the A.alpha., B.beta. and .gamma. sequences.

Southern blot analysis of transgenic mice prepared essentially as described above demonstrated that more than 50% of progeny contained all three fibrinogen sequences. Examination of milk from positive animals by reducing SDS polyacrylamide gel electrophoresis demonstrated the presence of all three protein chains at concentrations up to 1 mg/ml. The amount of fully assembled fibrinogen was related to the ratios of individual subunits present in the milk. No apparent phenotype was associated with high concentrations of human fibrinogen in mouse milk.

Example IV

Donor ewes are treated with an intravaginal progesterone-impregnated sponge (CHRONOGEST Goat Sponge, Intervet, Cambridge, UK) on day 0. Sponges are left in situ for ten or twelve days.

Superovulation is induced by treatment of donor ewes with a total of one unit of ovine follicle stimulating hormone (OFSH) (OVAGEN, Horizon Animal Reproduction Technology Pty. Ltd., New Zealand) administered in eight intramuscular injections of 0.125 units per injection starting at 5:00 pm on day -4 and ending at 8:00 am on day 0. Donors are injected intramuscularly with 0.5 ml of a luteolytic agent (ESTRUMATE, Vet-Drug) on day -4 to cause regression of the corpus luteum, to allow return to estrus and ovulation. To synchronize ovulation, the donor animals are injected intramuscularly with 2 ml of a synthetic releasing hormone analog (RECEPTAL, Vet-Drug) at 5:00 pm on day 0.

Donors are starved of food and water for at least 12 hours before artificial insemination (A.I.). The animals are artificially inseminated by intrauterine laparoscopy under sedation and local anesthesia on day 1. Either xylazine (ROMPUN, Vet-Drug) at a dose rate of 0.05-0.1 ml per 10 kg bodyweight or ACP injection 10 mg/ml (Vet-Drug) at a dose rate of 0.1 ml per 10 kg bodyweight is injected intramuscularly approximately fifteen minutes before A.I. to provide sedation. A.I. is carried out using freshly collected semen from a Poll Dorset ram. Semen is diluted with equal parts of filtered phosphate buffered saline, and 0.2 ml of the diluted semen is injected per uterine horn. Immediately pre- or post-A.I., donors are given an intramuscular injection of AMOXYPEN (Vet-Drug).

Fertilized eggs are recovered on day 2 following starvation of donors of food and water from 5:00 pm on day 1. Recovery is carried out under general anesthesia induced by an intravenous injection of 5% thiopentone sodium (INTRAVAL SODIUM, Vet-Drug) at a dose rate of 3 ml per 10 kg bodyweight. Anesthesia is maintained by inhalation of 1-2% Halothane/O.sub.2/N.sub.2O after intubation. To recover the fertilized eggs, a laparotomy incision is made, and the uterus is exteriorized. The eggs are recovered by retrograde flushing of the oviducts with Ovum Culture Medium (Advanced Protein Products, Brierly Hill, West Midlands, UK) supplemented with bovine serum albumin of New Zealand origin. After flushing, the uterus is returned to the abdomen, and the incision is closed. Donors are allowed to recover post-operatively or are euthanized. Donors that are allowed to recover are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately pre- or post-operatively.

Plasmids containing the three fibrinogen chain expression units are digested with Mlu I, and the expression unit fragments are recovered and purified on sucrose density gradients. The fragment concentrations are determined by fluorimetry and diluted in Dulbecco's phosphate buffered saline without calcium and magnesium as described above. The concentration is adjusted to 6 .mu.g/ml and approximately 2 pl of the mixture is microinjected into one pronucleus of each fertilized eggs with visible pronuclei.

All fertilized eggs surviving pronuclear microinjection are cultured in vitro at 38.5.degree. C. in an atmosphere of 5% CO.sub.2:5% O.sub.2:90% N.sub.2 and about .about.100% humidity in a bicarbonate buffered synthetic oviduct medium (see Table) supplemented with 20% v/v vasectomized ram serum. The serum may be heat inactivated at 56.degree. C. for 30 minutes and stored frozen at -20.degree. C. prior to use. The fertilized eggs are cultured for a suitable period of time to allow early embryo mortality (caused by the manipulation techniques) to occur. These dead or arrested embryos are discarded. Embryos having developed to 5 or 6 cell divisions are transferred to synchronized recipient ewes.

TABLE-US-00001 TABLE Synthetic Oviduct Medium Stock A (Lasts 3 Months) NaCl 6.29 g KCl 0.534 g KH.sub.2SO.sub.4 0.162 g MgSO.sub.4:7H.sub.2O 0.182 g Penicillin 0.06 g Sodium Lactate 60% syrup 0.6 mls Super H.sub.2O 99.4 mls Stock B (Lasts 2 weeks) NaHCO.sub.3 0.21 g Phenol red 0.001 g Super H.sub.2O 10 mls Stock C (Lasts 2 weeks) Sodium Pyruvate 0.051 g Super H.sub.2O 10 mls Stock D (Lasts 3 months) CaCl.sub.2.cndot.2H.sub.2O 0.262 g Super H.sub.2O 10 mls Stock E (Lasts 3 months) Hepes 0.651 g Phenol red 0.001 g Super H.sub.2O 10 mls To make up 10 mls of Bicarbonate Buffered Medium STOCK A 1 ml STOCK B 1 ml STOCK C 0.07 ml STOCK D 0.1 ml Super H.sub.2O 7.83 ml Osmolarity should be 265-285 mOsm. Add 2.5 ml of heat inactivated sheep serum and filter sterilize. To make up 10 mls HEPES Buffered Medium STOCK A 1 ml STOCK B 0.2 ml STOCK C 0.07 ml STOCK D 0.1 ml STOCK E 0.8 ml Super H.sub.2O 7.83 ml Osmolarity should be 265-285 mOsm. Add 2.5 ml of heat inactivated sheep serum and filter sterilize.

Recipient ewes are treated with an intravaginal progesterone-impregnated sponge (Chronogest Ewe Sponge or Chronogest Ewe-Lamb Sponge, Intervet) left in situ for 10 or 12 days. The ewes are injected intramuscularly with 1.5 ml (300 iu) of a follicle stimulating hormone substitute (P.M.S.G., Intervet) and with 0.5 ml of a luteolytic agent (Estrumate, Coopers Pitman-Moore) at sponge removal on day -1. The ewes are tested for estrus with a vasectomized ram between 8:00 am and 5:00 pm on days 0 and 1.

Embryos surviving in vitro culture are returned to recipients (starved from 5:00 pm on day 5 or 6) on day 6 or 7. Embryo transfer is carried out under general anesthesia as described above. The uterus is exteriorized via a laparotomy incision with or without laparoscopy. Embryos are returned to one or both uterine horns only in ewes with at least one suitable corpora lutea. After replacement of the uterus, the abdomen is closed, and the recipients are allowed to recover. The animals are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately pre- or post-operatively.

Lambs are identified by ear tags and left with their dams for rearing. Ewes and lambs are either housed and fed complete diet concentrates and other supplements and or ad lib. hay, or are let out to grass.

Within the first week of life (or as soon thereafter as possible without prejudicing health), each lamb is tested for the presence of the heterologous DNA by two sampling procedures. A 10 ml blood sample is taken from the jugular vein into an EDTA vacutainer. If fit enough, the lambs also have a second 10 ml blood sample taken within one week of the first. Tissue samples are taken by tail biopsy as soon as possible after the tail has become desensitized after the application of a rubber elastrator ring to its proximal third (usually within 200 minutes after "tailing"). The tissue is placed immediately in a solution of tail buffer. Tail samples are kept at room temperature and analyzed on the day of collection. All lambs are given an intramuscular injection of Amoxypen L.A. at the manufacturer's recommended dose rate immediately post-biopsy, and the cut end of the tail is sprayed with an antibiotic spray.

DNA is extracted from sheep blood by first separating white blood cells. A 10 ml sample of blood is diluted in 20 ml of Hank's buffered saline (HBS; obtained from Sigma Chemical Co.). Ten ml of the diluted blood is layered over 5 ml of Histopaque (Sigma) in each of two 15 ml screw-capped tubes. The tubes are centrifuged at 3000 rpm (2000.times.g max.), low brake for 15 minutes at room temperature. White cell interfaces are removed to a clean 15 ml tube and diluted to 15 ml in HBS. The diluted cells are spun at 3000 rpm for 10 minutes at room temperature, and the cell pellet is recovered and resuspended in 2-5 ml of tail buffer.

To extract DNA from the white cells, 10% SDS is added to the resuspended cells to a final concentration of 1%, and the tube is inverted to mix the solution. One mg of fresh proteinase K solution is added, and the mixture is incubated overnight at 45.degree. C. DNA is extracted using an equal volume of phenol/chloroform (.times.3) and chloroform/isoamyl alcohol (.times.1). The DNA is then precipitated by adding 0.1 volume of 3M NaOAc and 2 volumes of ethanol, and the tube is inverted to mix. The precipitated DNA is spooled out using a clean glass rod with a sealed end. The spool is washed in 70% ethanol, and the DNA is allowed to partially dry, then is redissolved in TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.4).

DNA samples from blood and tail are analyzed by Southern blotting using probes for the BLG promoter region and the fibrinogen chain coding regions.

From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

SEQUENCE LISTINGS

1

275943 base pairsnucleic aciddoublelinearDNA (genomic)Human Fibrinogen A-alpha chainCDS join(31..84, 1154..1279, 1739..1922, 3055..3200, 3786..5210) 1GTCTAGGAGC CAGCCCCACC CTTAGAAAAG ATG TTT TCC ATG AGG ATC GTC TGC 54 Met Phe Ser Met Arg Ile Val Cys 1 5CTA GTT CTA AGT GTG GTG GGC ACA GCA TGG GTATGGCCCT TTTCATTTTT 104Leu Val Leu Ser Val Val Gly Thr Ala Trp 10 15TCTTCTTGCT TTCTCTCTGG TGTTTATTCC ACAAAGAGCC TGGAGGTCAG AGTCTACCTG 164CTCTATGTCC TGACACACTC TTAGCTTTAT GACCCCAGGC CTGGGAGGAA ATTTCCTGGG 224TGGGCTTGAC ACCTCAAGAA TACAGGGTAA TATGACACCA AGAGGAAGAT CTTAGATGGA 284TGAGAGTGTA CAACTACAAG GGAAACTTTA GCATCTGTCA TTCAGTCTTA CCACATTTTG 344TTTTGTTTTG TTTTAAAAAG GGCAAGAATT ATTTGCCATC CTTGTACCTA TAAAGCCTTG 404GTGCATTATA ATGCTAGTTA ATGGAATAAA ACATTTTATG GTAAGATTTG TTTTCTTTAG 464TTATTAATTT CTTGCTACTT GTCCATAATA AGCAGAACTT TTAGTGTTAG TACAGTTTTG 524CTGAAAGGTT ATTGTTGTGT TTGTCAAGAC AGAAGAAAAA GCAAACGAAT TATCTTTGGA 584AATATCTTTG CAGTATCAGA AGAGATTAGT TAGTAAGGCA ATACGCTTTT CCGCAGTAAT 644GGTATTCTTT TAAATTATGA ATCCATCTCT AAAGGTTACA TAGAAACTTG AAGGAGAGAG 704GAACATTCAG TTAAGATAGT CTAGGTTTTT CTACTGAAGC AGCAATTACA GGAGAAAGAG 764CTCTACAGTA GTTTTCAACT TTCTGTCTGC AGTCATTAGT AAAAATGAAA AGGTAAAATT 824TAACTGATTT TATAGATTCA AATAATTTTC CTTTTAGGAT GGATTCTTTA AAACTCCTAA 884TATTTATCAA ATGCTTATTT AAGTGTCACA CACAGTTAAG AAATTTGTAC ACCTTGTCTC 944CTTTAATTCT CATAACAACT CCATAAAATG GGTCCTAGGA TTTCCATTTG AAGATAAGAA 1004ACCTGAAGCT TGCCGAAGCC CTGTGTCTGC TCTCCTTAAT CTCTGTGAGA GTGCCATCTC 1064TTCCTGGGGA CTTGTAGGCA TGCCACTGTC TCCTCTTCTG GCTAACATTG CTGTTGCTCT 1124CTTTTGTGTA TGTGAATGAA TCTTTAAAG ACT GCA GAT AGT GGT GAA GGT GAC 1177 Thr Ala Asp Ser Gly Glu Gly Asp 20 25TTT CTA GCT GAA GGA GGA GGC GTG CGT GGC CCA AGG GTT GTG GAA AGA 1225Phe Leu Ala Glu Gly Gly Gly Val Arg Gly Pro Arg Val Val Glu Arg 30 35 40CAT CAA TCT GCC TGC AAA GAT TCA GAC TGG CCC TTC TGC TCT GAT GAA 1273His Gln Ser Ala Cys Lys Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu 45 50 55GAC TGG GTAAGCAGTC AGCGGGGGAA GCAGGAGATT CCTTCCCTCT GATGCTAGAG 1329Asp Trp 60GGGCTCACAG GCTGACCTGA TTGGTCCCAG AAACTTTTTT AAATAGAAAA TAATTGAATA 1389GTTACCTACA TAGCAAATAA AGAAAAGGAA CCTACTCCCA AGAGCACTGT TTATTTACCT 1449CCCCAACTCT GGATCATTAG TGGGTGAACA GACAGGATTT CAGTTGCATG CTCAGGCAAA 1509ACCAGGCTCC TGAGTATTGT GGCCTCAATT TCCTGGCACC TATTTATGGC TAAGTGGACC 1569CTCATTCCAG AGTTTCTCTG CGACCTCTAA CTAGTCCTCT TACCTACTTT TAAGCCAACT 1629TATCTGGAAG AGAAAGGGTA GGAAGAAATG GGGGCTGCAT GGAAACATGC AAAATTATTC 1689TGAATCTGAG AGATAGATCC TTACTGTAAT TTTCTCCCTT CACTTTCAG AAC TAC 1744 Asn TyrAAA TGC CCT TCT GGC TGC AGG ATG AAA GGG TTG ATT GAT GAA GTC AAT 1792Lys Cys Pro Ser Gly Cys Arg Met Lys Gly Leu Ile Asp Glu Val Asn 65 70 75CAA GAT TTT ACA AAC AGA ATA AAT AAG CTC AAA AAT TCA CTA TTT GAA 1840Gln Asp Phe Thr Asn Arg Ile Asn Lys Leu Lys Asn Ser Leu Phe Glu 80 85 90TAT CAG AAG AAC AAT AAG GAT TCT CAT TCG TTG ACC ACT AAT ATA ATG 1888Tyr Gln Lys Asn Asn Lys Asp Ser His Ser Leu Thr Thr Asn Ile Met 95 100 105 110GAA ATT TTG AGA GGC GAT TTT TCC TCA GCC AAT A GTAAGTATTA 1932Glu Ile Leu Arg Gly Asp Phe Ser Ser Ala Asn 115 120CATATTTACT TCTTTGACTT TATAACAGAA ACAACAAAAA TCCTAAATAA ATATGATATC 1992CGCTTATATC TATGACAATT TCATCCCAAA GTACTTAGTG TAGAAACACA TACCTTCATA 2052ATATCCCTGA AAATTTTAAG AGGGAGCTTT TGTTTTCGTT ATTTTTTCAA AGTAAAAGAT 2112GTTAACTGAG ATTGTTTAAG GTCACAAAAT AAGTCAGAAT TTTGGATTAA AACAAGAATT 2172TAAATGTGTT CTTTTCAACA GTATATACTG AAAGTAGGAT GGGTCAGACT CTTTGAGTTG 2232ATATTTTTGT TTCTGCTTTG TAAAGGTGAA AACTGAGAGG TCAAGGAACT TGTTCAAAGA 2292CACAGAGCTG GGAATTCAAC TCCCAGACTC CACTGAGCTG ATTAGGTAGA TTTTTAAATT 2352TAAAATATAG GGTCAAGCTA CGTCATTCTC ACAGTCTACT CATTAGGGTT AGGAAACATT 2412GCATTCACTC TGGGCATGGA CAGCGAGTCT AGGGAGTCCT CAGTTTCTCA AGTTTTGCTT 2472TGCCTTTTTA CACCTTCACA AACACTTGAC ATTTAAAATC AGTGATGCCA ACACTAGCTG 2532GCAAGTGAGT GATCCTGTTG ACCCAAAACA GCTTAGGAAC CATTTCAAAT CTATAGAGTT 2592AAAAAGAAAA GCTCATCAGT AAGAAAATCC AATATGTTCA AGTCCCTTGA TTAAGGATGT 2652TATAAAATAA TTGAAATGCA ATCAAACCAA CTATTTTAAC TCCAAATTAC ACCTTTAAAA 2712TTCCAAAGAA AGTTCTTCTT CTATATTTCT TTGGGATTAC TAATTGCTAT TAGGACATCT 2772TAACTGGCAT TCATGGAAGG CTGCAGGGCA TAACATTATC CAAAAGTCAA ATGCCCCATA 2832GGTTTTGAAC TCACAGATTA AACTGTAACC AAAATAAAAT TAGGCATATT TACAAGCTAG 2892TTTCTTTCTT TCTTTTTTCT CTTTCTTTCT TTCTTTCTTT CTTTCTTTCT TTCTTTCTTT 2952CTTTCTTTCT TTCTCCTTCC TTCCTTTCTT CCTTTCTTTT TTGCTGGCAA TTACAGACAA 3012ATCACTCAGC AGCTACTTCA ATAACCATAT TTTCGATTTC AG AC CGT GAT AAT 3065 Asn Arg Asp Asn 125ACC TAC AAC CGA GTG TCA GAG GAT CTG AGA AGC AGA ATT GAA GTC CTG 3113Thr Tyr Asn Arg Val Ser Glu Asp Leu Arg Ser Arg Ile Glu Val Leu 130 135 140AAG CGC AAA GTC ATA GAA AAA GTA CAG CAT ATC CAG CTT CTG CAG AAA 3161Lys Arg Lys Val Ile Glu Lys Val Gln His Ile Gln Leu Leu Gln Lys 145 150 155AAT GTT AGA GCT CAG TTG GTT GAT ATG AAA CGA CTG GAG GTAAGTATGT 3210Asn Val Arg Ala Gln Leu Val Asp Met Lys Arg Leu Glu 160 165 170GGCTGTGGTC CCGAGTGTCC TTGTTTTTGA GTAGAGGGAA AAGGAAGGCG ATAGTTATGC 3270ACTGAGTGTC TACTATATGC AGAGAAAAGT GTTATATCCA TCATCTACCT AAAAGTAGGT 3330ATTATTTTCC TCACTCCACA GTTGAAGAAA AAAAAATTCA GAGATATTAA GTAAATTTTC 3390CAACGTACAT AGATAGTAAT TCAAAGCAAT GTTCAGTCCC TGTCTATTCC AAGCCATTAC 3450ATCACCACAC CTCTGAGCCC TCAGCCTGAG TTCACCAAGG ATCATTTAAT TAGCGTTTCC 3510TTTGAGAGGG AATAGCACCT TACTCTTGAT CCATTCTGAG GCTAAGATGA ATTAAACAGC 3570ATCCATTGCT TATCCTGGCT AGCCCTGCAA TACCCAACAT CTCTTCCACT GAGGGTGCTC 3630GATAGGCAGA AAACAGAGAA TATTAAGTGG TAGGTCTCCG AGTCAAAAAA AATGAAACCA 3690GTTTCCAGAA GGAAAATTAA CTACCAGGAA CTCAATAGAC GTAGTTTATG TATTTGTATC 3750TACATTTTCT CTTTATTTTT CTCCCCTCTC TCTAG GTG GAC ATT GAT ATT AAG 3803 Val Asp Ile Asp Ile Lys 175ATC CGA TCT TGT CGA GGG TCA TGC AGT AGG GCT TTA GCT CGT GAA GTA 3851Ile Arg Ser Cys Arg Gly Ser Cys Ser Arg Ala Leu Ala Arg Glu Val 180 185 190GAT CTG AAG GAC TAT GAA GAT CAG CAG AAG CAA CTT GAA CAG GTC ATT 3899Asp Leu Lys Asp Tyr Glu Asp Gln Gln Lys Gln Leu Glu Gln Val Ile 195 200 205GCC AAA GAC TTA CTT CCC TCT AGA GAT AGG CAA CAC TTA CCA CTG ATA 3947Ala Lys Asp Leu Leu Pro Ser Arg Asp Arg Gln His Leu Pro Leu Ile 210 215 220AAA ATG AAA CCA GTT CCA GAC TTG GTT CCC GGA AAT TTT AAG AGC CAG 3995Lys Met Lys Pro Val Pro Asp Leu Val Pro Gly Asn Phe Lys Ser Gln225 230 235 240CTT CAG AAG GTA CCC CCA GAG TGG AAG GCA TTA ACA GAC ATG CCG CAG 4043Leu Gln Lys Val Pro Pro Glu Trp Lys Ala Leu Thr Asp Met Pro Gln 245 250 255ATG AGA ATG GAG TTA GAG AGA CCT GGT GGA AAT GAG ATT ACT CGA GGA 4091Met Arg Met Glu Leu Glu Arg Pro Gly Gly Asn Glu Ile Thr Arg Gly 260 265 270GGC TCC ACC TCT TAT GGA ACC GGA TCA GAG ACG GAA AGC CCC AGG AAC 4139Gly Ser Thr Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro Arg Asn 275 280 285CCT AGC AGT GCT GGA AGC TGG AAC TCT GGG AGC TCT GGA CCT GGA AGT 4187Pro Ser Ser Ala Gly Ser Trp Asn Ser Gly Ser Ser Gly Pro Gly Ser 290 295 300ACT GGA AAC CGA AAC CCT GGG AGC TCT GGG ACT GGA GGG ACT GCA ACC 4235Thr Gly Asn Arg Asn Pro Gly Ser Ser Gly Thr Gly Gly Thr Ala Thr305 310 315 320TGG AAA CCT GGG AGC TCT GGA CCT GGA AGT GCT GGA AGC TGG AAC TCT 4283Trp Lys Pro Gly Ser Ser Gly Pro Gly Ser Ala Gly Ser Trp Asn Ser 325 330 335GGG AGC TCT GGA ACT GGA AGT ACT GGA AAC CAA AAC CCT GGG AGC CCT 4331Gly Ser Ser Gly Thr Gly Ser Thr Gly Asn Gln Asn Pro Gly Ser Pro 340 345 350AGA CCT GGT AGT ACC GGA ACC TGG AAT CCT GGC AGC TCT GAA CGC GGA 4379Arg Pro Gly Ser Thr Gly Thr Trp Asn Pro Gly Ser Ser Glu Arg Gly 355 360 365AGT GCT GGG CAC TGG ACC TCT GAG AGC TCT GTA TCT GGT AGT ACT GGA 4427Ser Ala Gly His Trp Thr Ser Glu Ser Ser Val Ser Gly Ser Thr Gly 370 375 380CAA TGG CAC TCT GAA TCT GGA AGT TTT AGG CCA GAT AGC CCA GGC TCT 4475Gln Trp His Ser Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser385 390 395 400GGG AAC GCG AGG CCT AAC AAC CCA GAC TGG GGC ACA TTT GAA GAG GTG 4523Gly Asn Ala Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe Glu Glu Val 405 410 415TCA GGA AAT GTA AGT CCA GGG ACA AGG AGA GAG TAC CAC ACA GAA AAA 4571Ser Gly Asn Val Ser Pro Gly Thr Arg Arg Glu Tyr His Thr Glu Lys 420 425 430CTG GTC ACT TCT AAA GGA GAT AAA GAG CTC AGG ACT GGT AAA GAG AAG 4619Leu Val Thr Ser Lys Gly Asp Lys Glu Leu Arg Thr Gly Lys Glu Lys 435 440 445GTC ACC TCT GGT AGC ACA ACC ACC ACG CGT CGT TCA TGC TCT AAA ACC 4667Val Thr Ser Gly Ser Thr Thr Thr Thr Arg Arg Ser Cys Ser Lys Thr 450 455 460GTT ACT AAG ACT GTT ATT GGT CCT GAT GGT CAC AAA GAA GTT ACC AAA 4715Val Thr Lys Thr Val Ile Gly Pro Asp Gly His Lys Glu Val Thr Lys465 470 475 480GAA GTG GTG ACC TCC GAA GAT GGT TCT GAC TGT CCC GAG GCA ATG GAT 4763Glu Val Val Thr Ser Glu Asp Gly Ser Asp Cys Pro Glu Ala Met Asp 485 490 495TTA GGC ACA TTG TCT GGC ATA GGT ACT CTG GAT GGG TTC CGC CAT AGG 4811Leu Gly Thr Leu Ser Gly Ile Gly Thr Leu Asp Gly Phe Arg His Arg 500 505 510CAC CCT GAT GAA GCT GCC TTC TTC GAC ACT GCC TCA ACT GGA AAA ACA 4859His Pro Asp Glu Ala Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr 515 520 525TTC CCA GGT TTC TTC TCA CCT ATG TTA GGA GAG TTT GTC AGT GAG ACT 4907Phe Pro Gly Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr 530 535 540GAG TCT AGG GGC TCA GAA TCT GGC ATC TTC ACA AAT ACA AAG GAA TCC 4955Glu Ser Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser545 550 555 560AGT TCT CAT CAC CCT GGG ATA GCT GAA TTC CCT TCC CGT GGT AAA TCT 5003Ser Ser His His Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly Lys Ser 565 570 575TCA AGT TAC AGC AAA CAA TTT ACT AGT AGC ACG AGT TAC AAC AGA GGA 5051Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly 580 585 590GAC TCC ACA TTT GAA AGC AAG AGC TAT AAA ATG GCA GAT GAG GCC GGA 5099Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met Ala Asp Glu Ala Gly 595 600 605AGT GAA GCC GAT CAT GAA GGA ACA CAT AGC ACC AAG AGA GGC CAT GCT 5147Ser Glu Ala Asp His Glu Gly Thr His Ser Thr Lys Arg Gly His Ala 610 615 620AAA TCT CGC CCT GTC AGA GGT ATC CAC ACT TCT CCT TTG GGG AAG CCT 5195Lys Ser Arg Pro Val Arg Gly Ile His Thr Ser Pro Leu Gly Lys Pro625 630 635 640TCC CTG TCC CCC TAGACTAAGT TAAATATTTC TGCACAGTGT TCCCATGGCC 5247Ser Leu Ser ProCCTTGCATTT CCTTCTTAAC TCTCTGTTAC ACGTCATTGA AACTACACTT TTTTGGTCTG 5307TTTTTGTGCT AGACTGTAAG TTCCTTGGGG GCAGGGCCTT TGTCTGTCTC ATCTCTGTAT 5367TCCCAAATGC CTAACAGTAC AGAGCCATGA CTCAATAAAT ACATGTTAAA TGGATGAATG 5427AATTCCTCTG AAACTCTATT TGAGCTTATT TAGTCAAATT CTTTCACTAT TCAAAGTGTG 5487TGCTATTAGA ATTGTCACCC AACTGATTAA TCACATTTTT AGTATGTGTC TCAGTTGACA 5547TTTAGGTCAG GCTAAATACA AGTTGTGTTA GTATTAAGTG AGCTTAGCTA CCTGTACTGG 5607TTACTTGCTA TTAGTTTGTG CAAGTAAAAT TCCAAATACA TTTGAGGAAA ATCCCCTTTG 5667CAATTTGTAG GTATAAATAA CCGCTTATTT GCATAAGTTC TATCCCACTG TAAGTGCATC 5727CTTTCCCTAT GGAGGGAAGG AAAGGAGGAA GAAAGAAAGG AAGGGAAAGA AACAGTATTT 5787GCCTTATTTA ATCTGAGCCG TGCCTATCTT TGTAAAGTTA AATGAGAATA ACTTCTTCCA 5847ACCAGCTTAA TTTTTTTTTT AGACTGTGAT GATGTCCTCC AAACACATCC TTCAGGTACC 5907CAAAGTGGCA TTTTCAATAT CAAGCTATCC GGATCC 5943644 amino acidsamino acidlinearprotein 2Met Phe Ser Met Arg Ile Val Cys Leu Val Leu Ser Val Val Gly Thr 1 5 10 15Ala Trp Thr Ala Asp Ser Gly Glu Gly Asp Phe Leu Ala Glu Gly Gly 20 25 30Gly Val Arg Gly Pro Arg Val Val Glu Arg His Gln Ser Ala Cys Lys 35 40 45Asp Ser Asp Trp Pro Phe Cys Ser Asp Glu Asp Trp Asn Tyr Lys Cys 50 55 60Pro Ser Gly Cys Arg Met Lys Gly Leu Ile Asp Glu Val Asn Gln Asp 65 70 75 80Phe Thr Asn Arg Ile Asn Lys Leu Lys Asn Ser Leu Phe Glu Tyr Gln 85 90 95Lys Asn Asn Lys Asp Ser His Ser Leu Thr Thr Asn Ile Met Glu Ile 100 105 110Leu Arg Gly Asp Phe Ser Ser Ala Asn Asn Arg Asp Asn Thr Tyr Asn 115 120 125Arg Val Ser Glu Asp Leu Arg Ser Arg Ile Glu Val Leu Lys Arg Lys 130 135 140Val Ile Glu Lys Val Gln His Ile Gln Leu Leu Gln Lys Asn Val Arg145 150 155 160Ala Gln Leu Val Asp Met Lys Arg Leu Glu Val Asp Ile Asp Ile Lys 165 170 175Ile Arg Ser Cys Arg Gly Ser Cys Ser Arg Ala Leu Ala Arg Glu Val 180 185 190Asp Leu Lys Asp Tyr Glu Asp Gln Gln Lys Gln Leu Glu Gln Val Ile 195 200 205Ala Lys Asp Leu Leu Pro Ser Arg Asp Arg Gln His Leu Pro Leu Ile 210 215 220Lys Met Lys Pro Val Pro Asp Leu Val Pro Gly Asn Phe Lys Ser Gln225 230 235 240Leu Gln Lys Val Pro Pro Glu Trp Lys Ala Leu Thr Asp Met Pro Gln 245 250 255Met Arg Met Glu Leu Glu Arg Pro Gly Gly Asn Glu Ile Thr Arg Gly 260 265 270Gly Ser Thr Ser Tyr Gly Thr Gly Ser Glu Thr Glu Ser Pro Arg Asn 275 280 285Pro Ser Ser Ala Gly Ser Trp Asn Ser Gly Ser Ser Gly Pro Gly Ser 290 295 300Thr Gly Asn Arg Asn Pro Gly Ser Ser Gly Thr Gly Gly Thr Ala Thr305 310 315 320Trp Lys Pro Gly Ser Ser Gly Pro Gly Ser Ala Gly Ser Trp Asn Ser 325 330 335Gly Ser Ser Gly Thr Gly Ser Thr Gly Asn Gln Asn Pro Gly Ser Pro 340 345 350Arg Pro Gly Ser Thr Gly Thr Trp Asn Pro Gly Ser Ser Glu Arg Gly 355 360 365Ser Ala Gly His Trp Thr Ser Glu Ser Ser Val Ser Gly Ser Thr Gly 370 375 380Gln Trp His Ser Glu Ser Gly Ser Phe Arg Pro Asp Ser Pro Gly Ser385 390 395 400Gly Asn Ala Arg Pro Asn Asn Pro Asp Trp Gly Thr Phe Glu Glu Val 405 410 415Ser Gly Asn Val Ser Pro Gly Thr Arg Arg Glu Tyr His Thr Glu Lys 420 425 430Leu Val Thr Ser Lys Gly Asp Lys Glu Leu Arg Thr Gly Lys Glu Lys 435 440 445Val Thr Ser Gly Ser Thr Thr Thr Thr Arg Arg Ser Cys Ser Lys Thr 450 455 460Val Thr Lys Thr Val Ile Gly Pro Asp Gly His Lys Glu Val Thr Lys465 470 475 480Glu Val Val Thr Ser Glu Asp Gly Ser Asp Cys Pro Glu Ala Met Asp 485 490 495Leu Gly Thr Leu Ser Gly Ile Gly Thr Leu Asp Gly Phe Arg His Arg 500 505 510His Pro Asp Glu Ala Ala Phe Phe Asp Thr Ala Ser Thr Gly Lys Thr 515 520 525Phe Pro Gly Phe Phe Ser Pro Met Leu Gly Glu Phe Val Ser Glu Thr 530 535 540Glu Ser Arg Gly Ser Glu Ser Gly Ile Phe Thr Asn Thr Lys Glu Ser545 550 555 560Ser Ser His His Pro Gly Ile Ala Glu Phe Pro Ser Arg Gly Lys Ser 565 570 575Ser Ser Tyr Ser Lys Gln Phe Thr Ser Ser Thr Ser Tyr Asn Arg Gly 580 585 590Asp Ser Thr Phe Glu Ser Lys Ser Tyr Lys Met Ala Asp Glu Ala Gly 595 600 605Ser Glu Ala Asp His Glu Gly Thr His Ser Thr Lys Arg Gly His Ala 610 615 620Lys Ser Arg Pro Val Arg Gly Ile His Thr Ser Pro Leu Gly Lys Pro625 630 635 640Ser Leu Ser Pro8878 base pairsnucleic aciddoublelinearDNA (genomic)human fibrinogen B-beta chainmisc_RNA 1..469exon 470..583intron 584..3257exon 3258..3449intron 3450..3938exon 3939..4122intron 4123..5042exon 5043..5270intron 5271..5830exon 5831..5944intron 5945..6632exon 6633..6758intron 6759..6966exon 6967..7252intron 7253..7870exon 7871..81023'UTR 8103..8537misc_RNA 8538..8878CDS

join(470..583, 3258..3449, 3939..4122, 5831..5944, 6633..6758, 6967..7252, 7871..8102) 3GAATTCATGC CCCTTTTGAA ATAGACTTAT GTCATTGTCA GAAAACATAA GCATTTATGG 60TATATCATTA ATGAGTCACG ATTTTAGTGG TTGCCTTGTG AGTAGGTCAA ATTTACTAAG 120CTTAGATTTG TTTTCTCACA TATTCTTTCG GAGCTTGTGT AGTTTCCACA TTAATTTACC 180AGAAACAAGA TACACACTCT CTTTGAGGAG TGCCCTAACT TCCCATCATT TTGTCCAATT 240AAATGAATTG AAGAAATTTA ATGTTTCTAA ACTAGACCAA CAAAGAATAA TAGTTGTATG 300ACAAGTAAAT AAGCTTTGCT GGGAAGATGT TGCTTAAATG ATAAAATGGT TCAGCCAACA 360AGTGAACCAA AAATTAAATA TTAACTAAGG AAAGGTAACC ATTTCTGAAG TCATTCCTAG 420CAGAGGACTC AGATATATAT AGGATTGAAG ATCTCTCAGT TAAGTCTAC ATG AAA 475 Met Lys 1AGG ATG GTT TCT TGG AGC TTC CAC AAA CTT AAA ACC ATG AAA CAT CTA 523Arg Met Val Ser Trp Ser Phe His Lys Leu Lys Thr Met Lys His Leu 5 10 15TTA TTG CTA CTA TTG TGT GTT TTT CTA GTT AAG TCC CAA GGT GTC AAC 571Leu Leu Leu Leu Leu Cys Val Phe Leu Val Lys Ser Gln Gly Val Asn 20 25 30GAC AAT GAG GAG GTGAATTTTT TAAAGCATTA TTATATTATT AGTAGTATTA 623Asp Asn Glu Glu 35TTAATATAAG ATGTAACATA ATCATATTAT GTGCTTATTT TAATGAAATT AGCATTGCTT 683ATAGTTATGA AATGGAATTG TTAACCTCTG ACTTATTGTA TTTAAAGAAT GTTTCATAGT 743ATTTCTTATA TAAAAACAAA GTAATTTCTT GTTTTCTAGT TTATCACCTT TGTTTTCTTA 803AGATGAGGAT GGCTTAGCTA ATGTAAGATG TGTTTTTCTC ACTTGCTATT CTGAGTACTG 863TGATTTTCAT TTACTTCTAG CAATACAGGA TTACAATTAA GAGGACAAGA TCTGAAAATC 923TCACAAACTA TAAAATAATA AAAGAGCAGA ATTTTAAGAT AAAAGAAACT GGTGGTAGGT 983AGATTGTTCT TTGGTGAAGG AAGGTAATAT ATATTGTTAC TGAGATTACT ATTTATAAAA 1043ATTATAACTA AGCCTAAAAG CAAAATACAT CAAGTGTAAT GATAGAAAAT GAAATATTGC 1103TTTTTTCAGA TGAAAAGTTC AAATTAGAGT TAGTGTGTAT TGTTATTATT AATAGTTATG 1163AAACACGGTT CAGTCTAATT TATTTATTTG TAGAACAGTT TGTCCTCAAC TATTATTTTT 1223GCTGACTTAT TGCTGTTAAT TTGCAGTTAC TAAAAATACA GAAATGCATT TAGGACAATG 1283GATATTTAAG AAATTTAAAT TTTATCATCA AACGTATCAT GGCCAAATTT CTTACATATA 1343GCATAGTATC ATTAAACTAG AAATAAGAAT ACACAATAAT ATTTAAATGA AGTGATTCAT 1403TTCGGATCAT TATTGAGTTT CAAGGGAACT TGAGTGTTGT ACTTATCAGA CTCTACATGT 1463AAGAACATAT AGTTAATCTG GTTGTGTGTG TAAAAACATA TGGTTAATCT GGTTAAGTCT 1523GGTTAATCAT ATTAGGTAAG AAAAATGTAA AGAATGTGTA AGACGAAATT TTTGTAAAGT 1583ACTCTGCAAA GCACTTTCAC ATTTCTGCTT ATCAACTAAA CCTCACAGAG ATAGTTTAAT 1643AGTTTAGGCT TTAAAATGGA TTTTGATTAT TCAACAAGTG GCCTTCATAA TTTCTTTAAG 1703TGTTTTTCTT TAAGTATATA CTTTCTTTAA ATATTTTTTA AAATTTCCTT TTCTCTAGTA 1763AAGCCAGACC ATCCATGCTA CCTCTCTAGT GGCACTCTGA AATAAAAAGA AAATAGTTTT 1823CTCTGTTATA ATTGTATTTG TAATAAGCAG ATGAATCACA TTTCTTAAAA TTTGTTTTAG 1883AGAGGGTAAG CTCTGACTAG GACCATGACT TCAATGTGAA ATATGTATAT ATCCTCCGAA 1943TCTTTACATA TTAAGAATGT ATATAGTCAA CTGGTTAAAC AGGAAAATCT GGAACAGCCT 2003GGCTGGGTTT TAATCTTAGC ACCATCCTAC TAAATGTTAA ATAATATTAT AATCTAATGA 2063ATAAATGACA ATGCAATTCC AAATAGAGTT CATCTGATGA CTTCTAGACT CACAAAATTG 2123CAAGAGAGCT CAGTTGTTGC TCAGTTGTTC CAAATCATGT CGTTTGTTAA TTTGTAATTA 2183AGCTCCAAAG GATGTATAGC TACTGACAAA AAAAAAAATG AGAATGTAGT TAATCCAAAT 2243CAAAACTTTC CTATTGCAAT GCGTATTTTC TGCTTCATTA TCCTTTAATA TAATATTTTA 2303AGTTAGCAAG TAATTTTAAT TACAATGCAC AAGCCTTGAG AATTATTTTA AATATAAGAA 2363AATCATAATG TTTGATAAAG AAATCATGTA AGAAATTTCA AGATAATGGT TTAACAAATA 2423ATTTTGTTGA TAGAAGATAA GACTAAAAGT GAAATTCGAA GTGGAGAGGA CACTTAAACT 2483GTAGTACTTG TTATGTGTGA TTCCAGTAAA AATAGTAATG AGCACTTATT ATTGCCAAGT 2543ACTGTTCTGA GGGTACCATA TGCAATAAGT TATTTAATCC TTACAATAAT CTTGTAAGGC 2603AGATTCAAAC TATCATTACA CTTATTTTAC AGATGAGAAA ACTGGGGCAC AGATAAAGCA 2663ACTTGCCCAA GGTCTCATAG CTGTAAGTCA ACCCTACGGT CAAGACCTAC AAGTAGCCGA 2723GCTCCAGAGT ACATTATGAG GGTCAAAGAT TGTCTTATTA CAAATAAATT CCAAGTAGAA 2783TCAACCTTTA ATAAGTCTTT AATGTCTCTT AAATATGTTT ATATAGGAGT CTAATCACCA 2843ATTCACAAAA ATGAAAGTAG GGAAATGATT AACAATAATC ATAGGAATCT AACAATCCAA 2903GTGGCTTGAG AATATTCATT CTTCTTGACA GTATAGATTC TTTACAATTT CGTAAGTTCC 2963AATGTATGTT TTAGGAATAT GAGGTCATTA CTATTCATAA TCTGATACAG CTTTATCCTA 3023AGGCCTCTCT TTAAAAACTA CACTGCATCA TAGCTTTTTT GTGCAGTTGG TCTTTCTACT 3083GTTACTGAAC AGTAAGCAAC CTACAGATTC ACTATCACCA ACCAGCCAGT TGATGGATCT 3143TAAGCAAATT ATCAAGCTTG TGATAACCTA AATTATAAAA TGAGGGTGTT GGAATAGTTA 3203CATTCCAAAT CTTCTATAAC ACTCTGTATT ATATTTCTGC CTCATTCCTT GTAG GGT 3260 GlyTTC TTC AGT GCC CGT GGT CAT CGA CCC CTT GAC AAG AAG AGA GAA GAG 3308Phe Phe Ser Ala Arg Gly His Arg Pro Leu Asp Lys Lys Arg Glu Glu 40 45 50 55GCT CCC AGC CTG AGG CCT GCC CCA CCG CCC ATC AGT GGA GGT GGC TAT 3356Ala Pro Ser Leu Arg Pro Ala Pro Pro Pro Ile Ser Gly Gly Gly Tyr 60 65 70CGG GCT CGT CCA GCC AAA GCA GCT GCC ACT CAA AAG AAA GTA GAA AGA 3404Arg Ala Arg Pro Ala Lys Ala Ala Ala Thr Gln Lys Lys Val Glu Arg 75 80 85AAA GCC CCT GAT GCT GGA GGC TGT CTT CAC GCT GAC CCA GAC CTG 3449Lys Ala Pro Asp Ala Gly Gly Cys Leu His Ala Asp Pro Asp Leu 90 95 100GTGGGTGCAC TGATGTTTCT TGCAGTGGTG GCTCTCTCAT GCAGAGAAAG CCTGTAGTCA 3509TGGCAGTCTG CTAATGTTTC ACTGACCCAC ATTACCATCA CTGTTATTTT GTTTGTTTAT 3569TTTGGAAATA AAATTCAAAA CATAAACATA TTGGGCCTTT GGTTTAGGCT TTCTTTCTTG 3629TTTTCTTTGG TCTGGGCCCA AAATTTCAAA TTAGGATATG TGGGTGCCAC CTTTCCATTT 3689GTATTTTGCC ACTGCCTTTG TTTAGTTGGT AAAATTTTCA TAGCCCAATT ATATTTTTTC 3749TGGGGTAAGT AATATTTTAA ATCTCTATGA GAGTATGATG ATGACTTTCG AATTTCTGGT 3809CTTACAGAAA ACCAAATAAT AAATTTTTAT GTTGGCTAAT CGTATCGCTG AATTTTCCTA 3869TGTGCTATTT TAACAAATGT CCATGACCCA AATCCTTCAT CTAATGCCTG CTATTTTCTT 3929TGTTTTTAG GGG GTG TTG TGT CCT ACA GGA TGT CAG TTG CAA GAG GCT 3977 Gly Val Leu Cys Pro Thr Gly Cys Gln Leu Gln Glu Ala 105 110 115TTG CTA CAA CAG GAA AGG CCA ATC AGA AAT AGT GTT GAT GAG TTA AAT 4025Leu Leu Gln Gln Glu Arg Pro Ile Arg Asn Ser Val Asp Glu Leu Asn 120 125 130AAC AAT GTG GAA GCT GTT TCC CAG ACC TCC TCT TCT TCC TTT CAG TAC 4073Asn Asn Val Glu Ala Val Ser Gln Thr Ser Ser Ser Ser Phe Gln Tyr 135 140 145ATG TAT TTG CTG AAA GAC CTG TGG CAA AAG AGG CAG AAG CAA GTA AAA G 4122Met Tyr Leu Leu Lys Asp Leu Trp Gln Lys Arg Gln Lys Gln Val Lys 150 155 160GTAGATATCC TTGTGCTTTC CATTCGATTT TCAGCTATAA AATTGGAACC GTTAGACTGC 4182CACGAGAATG CATGGTTGTG AGAAGATTAA CATTTCTGGG TTAGTGAATA GCATTCATAC 4242GCTTTTGGGC ACCTTCCCCT GCAACTTGCC AGATAAGCAC TATTCAGCTC TTATTCCCAG 4302TCTGACATCA GCAAGTGTGA TTTTCTATGA AAAATTCTAC TATGACTCCT TATTTTAAGT 4362ATACAAGAAA CTTGTGACTC AGAAGATAAT ATTTACAGAG TGGAAAAAAA CCCCTAGCAT 4422TTATAGTTTT AACATTTGAG GTTTTGAATG AGAGAGTTAT CCATAATATA TTCAATTGTG 4482TTGTGGATAA TGACACCTAA CCTGTGAATC TTGAGGTCAG AATGTTGAGT GCTGTTGACT 4542TGGTGGTCAG GAAACAGCTA GTGCGTGAGC CTGGCACAGG CATCTCAGTG AGTAGCATAC 4602CCACAGTTGG AAATTTTTCA AAGAAATCAA AGGAATCATG ACATCTTATA AATTTCAAGG 4662TTCTGCTATA CTTATGTGAA ATGGATAAAT AAATCAAGCA TATCCACTCT GTAAGATTGA 4722ACTTCTCAGA TGGAAGACCC CAATACTGCT TTCTCCTCTT TTCCCTCACC AAAGAAATAA 4782ACAACCTATT TCATTTATTA CTGGACACAA TCTTTAGCGT ATACCTATGG TAAATTACTA 4842GTATGGTGGT TAGGATTTAT GTTAATTTGT ATATGTCATG CGCCAAATCA TTTCCACTAA 4902ATATGACTAT ATATCATAAC TGCTTGGTGA TAGCTCAGTG TTTAATAGTT TATTCTCAGA 4962AAATCAAAAT TGTATAGTTA AATACATTAG TTTTATGAGG CAAAAATGCT AACTATTTCT 5022ACATAATTTC ATTTTTCCAG AT AAT GAA AAT GTA GTC AAT GAG TAC TCC 5071 Asp Asn Glu Asn Val Val Asn Glu Tyr Ser 165 170TCA GAA CTG GAA AAG CAC CAA TTA TAT ATA GAT GAG ACT GTG AAT AGC 5119Ser Glu Leu Glu Lys His Gln Leu Tyr Ile Asp Glu Thr Val Asn Ser 175 180 185AAT ATC CCA ACT AAC CTT CGT GTG CTT CGT TCA ATC CTG GAA AAC CTG 5167Asn Ile Pro Thr Asn Leu Arg Val Leu Arg Ser Ile Leu Glu Asn Leu190 195 200 205AGA AGC AAA ATA CAA AAG TTA GAA TCT GAT GTC TCA GCT CAA ATG GAA 5215Arg Ser Lys Ile Gln Lys Leu Glu Ser Asp Val Ser Ala Gln Met Glu 210 215 220TAT TGT CGC ACC CCA TGC ACT GTC AGT TGC AAT ATT CCT GTG GTG TCT 5263Tyr Cys Arg Thr Pro Cys Thr Val Ser Cys Asn Ile Pro Val Val Ser 225 230 235GGC AAA G GTAACTGATT CATAAACATA TTTTTAGAGA GTTCCAGAAG AACTCACACA 5320Gly LysCCAAAAATAA GAGAACAACA ACAACAACAA AAATGCTAAG TGGATTTTCC CAACAGATCA 5380TAATGACATT ACAGTACATC ATAAAAATAT CCTTAGCCAG TTGTGTTTTG GACTGGCCTG 5440GTGCATTTGC TGGTTTTGAT GAGCAGGATG GGGCACAGGT AGTCCCAGGG GTGGCTGATG 5500TGTGCATCTG CGTACTGGCT TGAACAGATG GCAGAACCAC AGATAGATGT AGAAGTTTCT 5560CCATTTTGTG TGTTCTGGGA GCTCATGGAT ATTCCAGGAC ACAAAAGGTG GAGAAGAGCT 5620TTGTTCATCC TCTTAGCAGA TAAACGTCCT CAAAACTGGG TTGGACTTAC TAAAGTAAAA 5680TGAAAATCTA ATATTTGTTA TATTATTTTC AAAGGTCTAT AATAACACAC TCCTTAGTAA 5740CTTATGTAAT GTTATTTTAA AGAATTGGTG ACTAAATACA AAGTAATTAT GTCATAAACC 5800CCTGAACATA ATGTTGTCTT ACATTTGCAG AA TGT GAG GAA ATT ATC AGG AAA 5853 Glu Cys Glu Glu Ile Ile Arg Lys 240 245GGA GGT GAA ACA TCT GAA ATG TAT CTC ATT CAA CCT GAC AGT TCT GTC 5901Gly Gly Glu Thr Ser Glu Met Tyr Leu Ile Gln Pro Asp Ser Ser Val 250 255 260AAA CCG TAT AGA GTA TAC TGT GAC ATG AAT ACA GAA AAT GGA G 5944Lys Pro Tyr Arg Val Tyr Cys Asp Met Asn Thr Glu Asn Gly 265 270 275GTAAGCTTTC GACAGTTGTT GACCTGTTGA TCTGTAATTA TTTGGATACC GTAAAATGCC 6004AGGAAACAAG GCCAGGTGTG GTGGCTCATA CCTGTAATTC CAGCACCTTG GGAGGCCAAA 6064GTGGGCTGAT AGCTTGAGCC TAGGAGTTTG AAACTAGCCT GGGCAACATA ATGAGACCCT 6124AACTCTACAA AAAAAAAAAA AATACCAAAA AAAAAAAAAA AATCAGCTGT GTTGGTAGTA 6184TGTGCCTGTA GTCCCAGCTA TCCAGGAGGC TGAGATGGGA GATCACCTGA GCCCACAACC 6244TGGAGTCTTG ATCATGCTAC TGAACTGTAG CCTGGGCAAC AGAGGATAGT GAGATCCTGT 6304CTCAAAAAAA AAAATTAATT AAAAAGCCAG GAAACAAGAC TTAGCTCTAA CATCTAACAT 6364AGCTGACAAA GGAGTAATTT GATGTGGAAT TCAACCTGAT ATTTAAAAGT TATAAAATAT 6424CTATAATTCA CAATTTGGGG TAAGATAAAG CACTTGCAGT TTCCAAAGAT TTTACAAGTT 6484TACCTCTCAT ATTTATTTCC TTATTGTGTC TATTTTAGAG CACCAAATAT ATACTAAATG 6544GAATGGACAG GGGATTCAGA TATTATTTTC AAAGTGACAT TATTTGCTGT TGGTTAATAT 6604ATGCTCTTTT TGTTTCTGTC AACCAAAG GA TGG ACA GTG ATT CAG AAC CGT 6655 Gly Trp Thr Val Ile Gln Asn Arg 280 285CAA GAC GGT AGT GTT GAC TTT GGC AGG AAA TGG GAT CCA TAT AAA CAG 6703Gln Asp Gly Ser Val Asp Phe Gly Arg Lys Trp Asp Pro Tyr Lys Gln 290 295 300GGA TTT GGA AAT GTT GCA ACC AAC ACA GAT GGG AAG AAT TAC TGT GGC 6751Gly Phe Gly Asn Val Ala Thr Asn Thr Asp Gly Lys Asn Tyr Cys Gly 305 310 315CTA CCA G GTAACGAACA GGCATGCAAA ATAAAATCAT TCTATTTGAA ATGGGATTTT 6808Leu ProTTTTAATTAA AAAACATTCA TTGTTGGAAG CCTGTTTTAG GCAGTTAAGA GGAGTTTCCT 6868GACAAAAATG TGGAAGCTAA AGATAAGGGA AGAAAGGCAG TTTTTAGTTT CCCAAAATTT 6928TATTTTTGGT GAGAGATTTT ATTTTGTTTT TCTTTTAG GT GAA TAT TGG CTT 6980 Gly Glu Tyr Trp Leu 320GGA AAT GAT AAA ATT AGC CAG CTT ACC AGG ATG GGA CCC ACA GAA CTT 7028Gly Asn Asp Lys Ile Ser Gln Leu Thr Arg Met Gly Pro Thr Glu Leu325 330 335 340TTG ATA GAA ATG GAG GAC TGG AAA GGA GAC AAA GTA AAG GCT CAC TAT 7076Leu Ile Glu Met Glu Asp Trp Lys Gly Asp Lys Val Lys Ala His Tyr 345 350 355GGA GGA TTC ACT GTA CAG AAT GAA GCC AAC AAA TAC CAG ATC TCA GTG 7124Gly Gly Phe Thr Val Gln Asn Glu Ala Asn Lys Tyr Gln Ile Ser Val 360 365 370AAC AAA TAC AGA GGA ACA GCC GGT AAT GCC CTC ATG GAT GGA GCA TCT 7172Asn Lys Tyr Arg Gly Thr Ala Gly Asn Ala Leu Met Asp Gly Ala Ser 375 380 385CAG CTG ATG GGA GAA AAC AGG ACC ATG ACC ATT CAC AAC GGC ATG TTC 7220Gln Leu Met Gly Glu Asn Arg Thr Met Thr Ile His Asn Gly Met Phe 390 395 400TTC AGC ACG TAT GAC AGA GAC AAT GAC GGC TG GTATGTGTGG 7262Phe Ser Thr Tyr Asp Arg Asp Asn Asp Gly Trp405 410 415CACTCTTTGC TCCTGCTTTA AAAATCACAC TAATATCATT ACTCAGAATC ATTAACAATA 7322TTTTTAATAG CTACCACTTC CTGGGCACTT ACTGTCAGCC ACTGTCCTAA GCTCTTTATG 7382CATCACTCGA AAGCATTTCA ACTATAAGGT AGACATTCTT ATTCTCATTT TACAGATGAG 7442ATTTAGAGAG ATTACGTGAT TTGTCCAATG TCACACAACT ACCCAGAGAT AAAACTAGAA 7502TTTGAGCACA GTTACTTTCT GAATAATGAG CATTTAGATA AATACCTATA TCTCTATATT 7562CTAAAGTGTG TGTGAAAACT TTCATTTTCA TTTCCAGGGT TCTCTGATAC TAAGGGTTGT 7622AAAAGCTATT ATTCCAGTAT AAAGTAACAA ACACAGTCCC TAGATGGATT GCCACAAAGG 7682CCCAGTTATC TCTCTTTCTT GCTATAGGGC ACAGGAGGTC TTTGGTGTAT TAGTGTGACT 7742CTATGTATAG CACCCAAAGG AAAGACTACT GTGCACACGA GTGTAGCAGT CTTTTATGGG 7802TAATCTGCAA AACGTAACTT GACCACCGTA GTTCTGTTTC TAATAACGCC AAACACATTT 7862TCTTTCAG G TTA ACA TCA GAT CCC AGA AAA CAG TGT TCT AAA GAA GAC 7910 Leu Thr Ser Asp Pro Arg Lys Gln Cys Ser Lys Glu Asp 420 425GGT GGT GGA TGG TGG TAT AAT AGA TGT CAT GCA GCC AAT CCA AAC GGC 7958Gly Gly Gly Trp Trp Tyr Asn Arg Cys His Ala Ala Asn Pro Asn Gly 430 435 440AGA TAC TAC TGG GGT GGA CAG TAC ACC TGG GAC ATG GCA AAG CAT GGC 8006Arg Tyr Tyr Trp Gly Gly Gln Tyr Thr Trp Asp Met Ala Lys His Gly445 450 455 460ACA GAT GAT GGT GTA GTA TGG ATG AAT TGG AAG GGG TCA TGG TAC TCA 8054Thr Asp Asp Gly Val Val Trp Met Asn Trp Lys Gly Ser Trp Tyr Ser 465 470 475ATG AGG AAG ATG AGT ATG AAG ATC AGG CCC TTC TTC CCA CAG CAA 8099Met Arg Lys Met Ser Met Lys Ile Arg Pro Phe Phe Pro Gln Gln 480 485 490TAGTCCCCAA TACGTAGATT TTTGCTCTTC TGTATGTGAC AACATTTTTG TACATTATGT 8159TATTGGAATT TTCTTTCATA CATTATATTC CTCTAAAACT CTCAAGCAGA CGTGAGTGTG 8219ACTTTTTGAA AAAAGTATAG GATAAATTAC ATTAAAATAG CACATGATTT TCTTTTGTTT 8279TCTTCATTTC TCTTGCTCAC CCAAGAAGTA ACAAAAGTAT AGTTTTGACA GAGTTGGTGT 8339TCATAATTTC AGTTCTAGTT GATTGCGAGA ATTTTCAAAT AAGGAAGAGG GGTCTTTTAT 8399CCTTGTCGTA GGAAAACCAT GACGGAAAGG AAAAACTGAT GTTTAAAAGT CCACTTTTAA 8459AACTATATTT ATTTATGTAG GATCTGTCAA AGAAAACTTC CAAAAAGATT TATTAATTAA 8519ACCAGACTCT GTTGCAATAA GTTAATGTTT TCTTGTTTTG TAATCCACAC ATTCAATGAG 8579TTAGGCTTTG CACTTGTAAG GAAGGAGAAG CGTTCACAAC CTCAAATAGC TAATAAACCG 8639GTCTTGAATA TTTGAAGATT TAAAATCTGA CTCTAGGACG GGCACGGTGG CTCACGACTA 8699TAATCCCAAC ACTTTGGGAG GCTGAGGCGG GCGGTCACAA GGTCAGGAGT TCAAGACCAG 8759CCTGACCAAT ATGGTGAAAC CCCATCTCTA CTAAAAATAC AAAAATTAGC CAGGCGTGGT 8819GGCAGGTGCC TGTAGGTCCC AGCTAGCCTG TGAGGTGGAG ATTGCATTGA GCCAAGATC 8878491 amino acidsamino acidlinearprotein 4Met Lys Arg Met Val Ser Trp Ser Phe His Lys Leu Lys Thr Met Lys 1 5 10 15His Leu Leu Leu Leu Leu Leu Cys Val Phe Leu Val Lys Ser Gln Gly 20 25 30Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg Gly His Arg Pro 35 40 45Leu Asp Lys Lys Arg Glu Glu Ala Pro Ser Leu Arg Pro Ala Pro Pro 50 55 60Pro Ile Ser Gly Gly Gly Tyr Arg Ala Arg Pro Ala Lys Ala Ala Ala 65 70 75 80Thr Gln Lys Lys Val Glu Arg Lys Ala Pro Asp Ala Gly Gly Cys Leu 85 90 95His Ala Asp Pro Asp Leu Gly Val Leu Cys Pro Thr Gly Cys Gln Leu 100 105 110Gln Glu Ala Leu Leu Gln Gln Glu Arg Pro Ile Arg Asn Ser Val Asp 115 120 125Glu Leu Asn Asn Asn Val Glu Ala Val Ser Gln Thr Ser Ser Ser Ser 130 135 140Phe Gln Tyr Met Tyr Leu Leu Lys Asp Leu Trp Gln Lys Arg Gln Lys145 150 155 160Gln Val Lys Asp Asn Glu Asn Val Val Asn Glu Tyr Ser Ser Glu Leu 165 170 175Glu Lys His Gln Leu Tyr Ile Asp Glu Thr Val Asn Ser Asn Ile Pro 180 185 190Thr Asn Leu Arg Val Leu Arg Ser Ile Leu Glu Asn Leu Arg Ser Lys 195 200 205Ile Gln Lys Leu Glu Ser Asp Val Ser Ala Gln Met Glu Tyr Cys Arg 210 215 220Thr Pro Cys Thr Val Ser Cys Asn Ile Pro Val Val Ser Gly Lys Glu225 230 235 240Cys Glu Glu Ile Ile Arg Lys Gly Gly Glu Thr Ser Glu Met Tyr Leu 245 250 255Ile Gln Pro Asp Ser Ser Val Lys Pro Tyr Arg Val Tyr Cys Asp Met 260 265 270Asn Thr Glu Asn Gly Gly Trp Thr Val Ile Gln Asn Arg Gln Asp Gly 275 280 285Ser Val Asp Phe Gly Arg Lys Trp Asp Pro Tyr Lys Gln Gly Phe Gly 290 295 300Asn Val Ala Thr Asn Thr Asp Gly Lys Asn Tyr Cys Gly Leu Pro Gly305 310 315 320Glu Tyr Trp Leu Gly Asn Asp Lys Ile Ser Gln Leu Thr Arg Met Gly 325 330 335Pro Thr Glu Leu Leu Ile Glu Met Glu Asp Trp Lys Gly Asp Lys Val 340 345 350Lys Ala His Tyr Gly Gly Phe Thr Val Gln Asn Glu Ala Asn Lys Tyr 355 360 365Gln Ile Ser Val Asn Lys Tyr Arg Gly Thr Ala Gly Asn Ala Leu Met 370

375 380Asp Gly Ala Ser Gln Leu Met Gly Glu Asn Arg Thr Met Thr Ile His385 390 395 400Asn Gly Met Phe Phe Ser Thr Tyr Asp Arg Asp Asn Asp Gly Trp Leu 405 410 415Thr Ser Asp Pro Arg Lys Gln Cys Ser Lys Glu Asp Gly Gly Gly Trp 420 425 430Trp Tyr Asn Arg Cys His Ala Ala Asn Pro Asn Gly Arg Tyr Tyr Trp 435 440 445Gly Gly Gln Tyr Thr Trp Asp Met Ala Lys His Gly Thr Asp Asp Gly 450 455 460Val Val Trp Met Asn Trp Lys Gly Ser Trp Tyr Ser Met Arg Lys Met465 470 475 480Ser Met Lys Ile Arg Pro Phe Phe Pro Gln Gln 485 49010564 base pairsnucleic aciddoublelinearDNA (genomic)human fibrinogen gamma chainCDS join(1799..1876, 1973..2017, 2207..2390, 2510 ..2603, 4211..4341, 4645..4778, 5758..5942, 7426 ..7703, 9342..9571) 5CTACACACTT CTTGAAGGCA AAGGCAATGC TGAAGTCACC TTTCATGTTC AAATCATATT 60AAAAAGTTAG CAAGATGTAA TTATCAGTGT ACTATGTAAA TCTTTGTGAA TGATCAATAA 120TTACATATTT TCATTATATA TATTTTAGTA GATAATATTT ATATACATTC AACATTCTAA 180ATATAGAAAG TTTACAGAGA AAAATAAAGC CTTTTTTTCC AATCCTGTCC TCCACCTCTG 240CATCCCATTC TTCTTCACAG AGGCAACTGA TTCAAGTCAT TACATAGTTA TTGAGTGTTA 300ACTACAACTA TGTTAAGTAC AGCTATATAT GTTAGATGCC GTAGCCACAG AAATCAGTTT 360ACAATCTAAT GCAGTGGATA CAGCATGTAT ACATATAATA TAAGGTTGCT ACAAATGCTA 420TCTGAGGTAG AGCTGTTTGA AAGAATACTA ATACTTAAAT GTTTAATTCA ACTGACTTGA 480TTGACAACTG ATTAGCTGAG TGGAAAAGAT GGATGAGAAA GATTGTGAGA CTTAATTGGC 540TGGTGGTATG GTGATATGAT TGACAATAAC TGCTAAGTCA GAGAGGGATA TATTAAGGAG 600GAGAAGAAAA GCAACAAATC TGGTTTTGAT GTGTTCACTT TGTTATAATT ATTGATTATT 660TACTGAATAT GAATATTTAT CTTTGTTTTT GAGTCAATAA ATATACCTTT GTAAAGACAG 720AATTAAAGTA TTAGTATTTC TTTCAAACTG GAGGCATTTC TCCCACTAAC ATATTTCATC 780AAAACTTATA ATAAGCTTGG TTCCAGAGGA AGAAATGAGG GATAACCAAA AATAGAGACA 840TTAATAATAG TGTAACGCCC AGTGATAAAT CTCAATAGGC AGTGATGACA GACATGTTTT 900CCCAAACACA AGGATGCTGT AAGGGCCAAA CAGAAATGAT GGCCCCTCCC CAGCACCTCA 960TTTTGCCCCT TCCTTCAGCT ATGCCTCTAC TCTCCTTTAG ATACAAGGGA GGTGGATTTT 1020TCTCTTCTCT GAGATAGCTT GATGGAACCA CAGGAACAAT GAAGTGGGCT CCTGGCTCTT 1080TTCTCTGTGG CAGATGGGGT GCCATGCCCA CCTTCAGACA AAGGGAAGAT TGAGCTCAAA 1140AGCTCCCTGA GAAGTGAGAG CCTATGAACA TGGTTGACAC AGAGGGACAG GAATGTATTT 1200CCAGGGTCAT TCATTCCTGG GAATAGTGAA CTGGGACATG GGGGAAGTCA GTCTCCTCCT 1260GCCACAGCCA CAGATTAAAA ATAATAATGT TAACTGATCC CTAGGCTAAA ATAATAGTGT 1320TAACTGATCC CTAAGCTAAG AAAGTTCTTT TGGTAATTCA GGTGATGGCA GCAGGACCCA 1380TCTTAAGGAT AGACTAGGTT TGCTTAGTTC GAGGTCATAT CTGTTTGCTC TCAGCCATGT 1440ACTGGAAGAA GTTGCATCAC ACAGCCTCCA GGACTGCCCT CCTCCTCACA GCAATGGATA 1500ATGCTTCACT AGCCTTTGCA GATAATTTTG GATCAGAGAA AAAACCTTGA GCTGGGCCAA 1560AAAGGAGGAG CTTCAACCTG TGTGCAAAAT CTGGGAACCT GACAGTATAG GTTGGGGGCC 1620AGGATGAGGA AAAAGGAACG GGAAAGACCT GCCCACCCTT CTGGTAAGGA GGCCCCGTGA 1680TCAGCTCCAG CCATTTGCAG TCCTGGCTAT CCCAGGAGCT TACATAAAGG GACAATTGGA 1740GCCTGAGAGG TGACAGTGCT GACACTACAA GGCTCGGAGC TCCGGGCACT CAGACATC 1798ATG AGT TGG TCC TTG CAC CCC CGG AAT TTA ATT CTC TAC TTC TAT GCT 1846Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile Leu Tyr Phe Tyr Ala 1 5 10 15CTT TTA TTT CTC TCT TCA ACA TGT GTA GCA GTAAGTGTGC TCTTCACAAA 1896Leu Leu Phe Leu Ser Ser Thr Cys Val Ala 20 25ACGTTGTTTA AAATGGAAAG CTGGAAAATA AAACAGATAA TAAACTAGTG AAATTTTCGT 1956ATTTTTTCTC TTTTAG TAT GTT GCT ACC AGA GAC AAC TGC TGC ATC TTA 2005 Tyr Val Ala Thr Arg Asp Asn Cys Cys Ile Leu 30 35GAT GAA AGA TTC GTAAGTAGTT TTTATGTTTC TCCCTTTGTG TGTGAACTGG 2057Asp Glu Arg Phe 40AGAGGGGCAG AGGAATAGAA ATAATTCCCT CATAAATATC ATCTGGCACT TGTAACTTTT 2117TAAAAACATA GTCTAGGTTT TACCTATTTT TCTTAATAGA TTTTAAGAGT AGCATCTGTC 2177TACATTTTTA ATCACTGTTA TATTTTCAG GGT AGT TAT TGT CCA ACT ACC TGT 2230 Gly Ser Tyr Cys Pro Thr Thr Cys 45GGC ATT GCA GAT TTC CTG TCT ACT TAT CAA ACC AAA GTA GAC AAG GAT 2278Gly Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys Asp 50 55 60 65CTA CAG TCT TTG GAA GAC ATC TTA CAT CAA GTT GAA AAC AAA ACA TCA 2326Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val Glu Asn Lys Thr Ser 70 75 80GAA GTC AAA CAG CTG ATA AAA GCA ATC CAA CTC ACT TAT AAT CCT GAT 2374Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro Asp 85 90 95GAA TCA TCA AAA CCA A GTGAGAAAAT AAAGACTACT GACCAAAAAA 2420Glu Ser Ser Lys Pro 100TAATAATAAT AATCTGTGAA GTTCTTTTGC TGTTGTTTTA GTTGTTCTAT TTGCTTAAGG 2480ATTTTTATGT CTCTGATCCT ATATTACAG AT ATG ATA GAC GCT GCT ACT TTG 2532 Asn Met Ile Asp Ala Ala Thr Leu 105 110AAG TCC AGG ATA ATG TTA GAA GAA ATT ATG AAA TAT GAA GCA TCG ATT 2580Lys Ser Arg Ile Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile 115 120 125TTA ACA CAT GAC TCA AGT ATT CG GTAAGGATTT TTGTTTTAAT TTGCTCTGCA 2633Leu Thr His Asp Ser Ser Ile Arg 130AGACTGATTT AGTTTTTATT TAATATTCTA TACTTGAGTG AAAGTAATTT TTAATGTGTT 2693TTCCCCATTT ATAATATCCC AGTGACATTA TGCCTGATTA TGTTGAGCAT AGTAGAGATA 2753GAAGTTTTTA GTGCAATATA AATTATACTG GGTTATAATT GCTTATTAAT AATCACATTG 2813AAGAAAGATG TTCTAGATGT CTTCAAATGC TAGTTTGACC ATATTTATCA AAAATTTTTT 2873CCCCATCCCC CATTTATCTT ACAACATAAA ATCAATCTCA TAGGAATTTG GGTGTTGAAA 2933ATAAAATCCT CTTTATAAAA ATGCTGACAA ATTGGTGGTT AAAAAAATTA GCAAGCAGAG 2993GCATAGTAAG GATTTTGGCT CCTAAAGTAA ATTATATTGA ATGTGGAGCA GGAAGAAACA 3053TGTCTTGAGA GACTAAGTGT GGCAAATATT GCAAAGCTCA TATTGATCAT TGCAGAATGA 3113ACCTGCATAG TCTCTTCCCT TCATTTGGAA GTGAATGTCT CTGTTAAAGC TTCTCAGGGA 3173CTCATAAACT TTCTGAACAT AAGGTCTCAG ATACAGTTTT AATATTTTTC CCCAATTTTT 3233TTTTCTGAAT TTTTCTCAAA GCAGCTTGAG AAATTGAGAT AAATAGTAGC TAGGGAGAAG 3293TGGCCCAGGA AAGATTTCTC CTCTTTTTGC TATCAGAGGG CCCTTGTTAT TATTGTTATT 3353ATTATTACTT GCATTATTAT TGTCCATCAT TGAAGTTGAA GGAGGTTATT GTACAGAAAT 3413TGCCTAAGAC AAGGTAGAGG GAAAACGTGG ACAAATAGTT TGTCTACCCT TTTTTACTTC 3473AAAGAAAGAA CGGTTTATGC ATTGTAGACA GTTTTCTATC ATTTTTGGAT ATTTGCAAGC 3533CACCCTGTAA GTAACTACAA AAGGAGGGTT TTTACTTCCC CCAGTCCATT CCCAAAGCTA 3593TGTAACCAGA AGCATTAAAG AAGAAAGGGG AAGTATCTGT TGTTTTATTT TACATACAAT 3653AACGTTCCAG ATCATGTCCC TGTGTAAGTT ATATTTTAGA TTGAAGCTTA TATGTATAGC 3713CTCAGTAGAT CCACAAGTGA AAGGTATACT CCTTCAGCAC ATGTGAATTA CTGAACTGAG 3773CTTTTCCTGC TTCTAAAGCA TCAGGGGGTG TTCCTATTAA CCAGTCTCGC CACTCTTGCA 3833GGTTGCTATC TGCTGTCCCT TATGCATAAA GTAAAAAGCA AAATGTCAAT GACATTTGCT 3893TATTGACAAG GACTTTGTTA TTTGTGTTGG GAGTTGAGAC AATATGCCCC ATTCTAAGTA 3953AAAAGATTCA GGTCCACATT GTATTCCTGT TTTAATTGAT TTTTTGATTT GTTTTTCTTT 4013TTCAAAAAGT TTATAATTTT AATTCATGTT AATTTAGTAA TATAATTTTA CATTTTCCTC 4073AAGAATGGAA TAATTTATCA GAAAGCACTT CTTAAGAAAA TACTTAGCAG TTTCCAAAGA 4133AAATATAAAA TTACTCTTCT GAAAGGAATA CTTATTTTTG TCTTCTTATT TTTGTTATCT 4193TATGTTTCTG TTTGTAG A TAT TTG CAG GAA ATA TAT AAT TCA AAT AAT CAA 4244 Tyr Leu Gln Glu Ile Tyr Asn Ser Asn Asn Gln 135 140 145AAG ATT GTT AAC CTG AAA GAG AAG GTA GCC CAG CTT GAA GCA CAG TGC 4292Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln Leu Glu Ala Gln Cys 150 155 160CAG GAA CCT TGC AAA GAC ACG GTG CAA ATC CAT GAT ATC ACT GGG AAA G 4341Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly Lys 165 170 175GTAACTGATG AAGGTTATAT TGGGATTAGG TTCATCAAAG TAAGTAATGT AAAGGAGAAA 4401GTATGTACTG GAAAGTATAG GAATAGTTTA GAAAGTGGCT ACCCATTAAG TCTAAGAATT 4461TCAGTTGTCT AGACCTTTCT TGAATAGCTA AAAAAAACAG TTTAAAAGGA ATGCTGATGT 4521GAAAAGTAAG AAAATTATTC TTGGAAAATG AATAGTTTAC TACATGTTAA AAGCTATTTT 4581TCAAGGCTGG CACAGTCTTA CCTGCATTTC AAACCACAGT AAAAGTCGAT TCTCCTTCTC 4641TAG AT TGT CAA GAC ATT GCC AAT AAG GGA GCT AAA CAG AGC GGG CTT 4688 Asp Cys Gln Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu 180 185 190TAC TTT ATT AAA CCT CTG AAA GCT AAC CAG CAA TTC TTA GTC TAC TGT 4736Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val Tyr Cys 195 200 205GAA ATC GAT GGG TCT GGA AAT GGA TGG ACT GTG TTT CAG AAG 4778Glu Ile Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys 210 215 220GTAATTTTTT CCCCACCATG TGTATTTAAT AAATTCCTAC ATTGTTTCTG CCATATGGCA 4838GATACTTTTC TAAGCACCTT GTGAACCGTA GCTCATTTAA TCCTTGCAAT AGCCCTAAGA 4898GGAAGGTACT TCTGTTACTC CTATTTACAG AAAAGGAAAC TGAGGCACAC AAGGTTAAAT 4958AACTTGCCCA AGACCACATA ACTAATAAGC AACAGAGTCA GCATTTGAAC CTAGGCAGTA 5018TAGTTTCAGA GTTTGTGACT TGACTCTATA TTGTACTGGC ACTGACTTTG TAGATTCATG 5078GTGGCACATA ATCATAGTAC CACAGTGACA AATAAAAAGA AGGAAACTCT TTTGTCAGGT 5138AGGTCAAGAC CTGAGGTTTC CCATCACAAG ATGAGGAAGC CCAACACCAC CCCCCACCAC 5198CCCACCACCA TCACCACCCT TTCACACACC AGAGGATACA CTTGGGCTGC TCCAAGACAA 5258GGAACCTGTG TTGCATCTGC CACTTGCTGA TACCCACTAG GAATCTTGGC TCCTTTACTT 5318TCTGTTTACC TCCCACCACT GTTATAACTG TTTCTACAGG GGGCGCTCAG AGGGAATGAA 5378TGGTGGAAGC ATTAGTTGCC AGACACCGAT TGAGCAATGG GTTCCATCAT AAGTGTAAGA 5438ATCAGTAATA TCCAGCTAGA GTTCTGAAGT CGTCTAGGTG TCTTTTTAAT ATTACCACTC 5498ATTTAGAATT TATGATGTGC CAGAAACCCT CTTAAGTATT TCTCTTATAT TCTCTCTCAT 5558GATCCTTGCA GCAACCCTAA GAAGTAACCA TCATTTTTCC TATTTGATAC ATGAGGAAAC 5618TGAGGTAGCT TGGCCAAGAT CACTTAGTTG GGAGTTGATA GAACCAGTGC TCTGTATTTT 5678TGACAAAATG TTGACAGCAT TCTCTTTACA TGCATTGATA GTCTATTTTC TCCTTTTGCT 5738CTTGCAAATG TGTAATTAG AGA CTT GAT GGC AGT GTA GAT TTC AAG AAA AAC 5790 Arg Leu Asp Gly Ser Val Asp Phe Lys Lys Asn 225 230TGG ATT CAA TAT AAA GAA GGA TTT GGA CAT CTG TCT CCT ACT GGC ACA 5838Trp Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Ser Pro Thr Gly Thr 235 240 245ACA GAA TTT TGG CTG GGA AAT GAG AAG ATT CAT TTG ATA AGC ACA CAG 5886Thr Glu Phe Trp Leu Gly Asn Glu Lys Ile His Leu Ile Ser Thr Gln250 255 260 265TCT GCC ATC CCA TAT GCA TTA AGA GTG GAA CTG GAA GAC TGG AAT GGC 5934Ser Ala Ile Pro Tyr Ala Leu Arg Val Glu Leu Glu Asp Trp Asn Gly 270 275 280AGA ACC AG GTACTGTTTT GAAATGACTT CCAACTTTTT ATTGTAAAGA 5982Arg Thr SerTTGCCTGGAA TGTGCACTTT CCAACTATCA ATAGACAATG GCAAATGCAG CCTGACAAAT 6042GCAAACAGCA CATCCAGCCA CCATTTTCTC CAGGAGTCTG TTTGGTTCTT GGGCAATCCA 6102AAAAGGTAAA TTCTATTCAG GATGAATCTA AGTGTATTGG TACAATCTAA TTACCCTGGA 6162ACCATTCAGA GTAATAGCTA ATTACTGAAC TTTTAATCAG TCCCAGGAAT TGAGCATAAA 6222ATTATAATTT TATCTAGTCT AAATTACTAT TTCATGAAGC AGGTATTATT ATTAATCCCA 6282TTTTATAGAT TAACTTGCTC AAAGTCACAT TGCTGATAAG TGGTAGAGGT AGAATTCAGA 6342CTCAAGTAGT TTAACTTTAG AGCCTGTCCT CTTAACAACT ATCCTGGTTG AAAAGCAAAT 6402ACAGCCTCTT CAGACTTCTC AGTGCCTTGA TGGCCATTTA TTCTGTCAAA TCATGAGCTA 6462CCCTAAAAGT AAACCAGCTA GCTCTTTTGA TGATCTAGAG GCTTCTTTTT GCTTGAGATA 6522TTTGAAGGTT TTAAGCATTG TTACCTAATT AAAATGCAGA AAAATATCCA ACCCTCTTGT 6582TATGTTTAAG GAATAGTGAA ATATATTGTC TTCAAACACA TGGACTTTTT TTTATTGCTT 6642GGTTGGTTTT TAATCCAGAA AGTGCTATAG TCAGTAGACC TTCTTCTAGG AAAGGACCTT 6702CCATTTCCCA GCCACTGGAG ATTAGAAAAT AAGCTAAATA TTTTCTGGAA ATTTCTGTTC 6762ATTCATTAAG GCCCATCCTT TCCCCCACTC TATAGAAGTG TTGTCCACTT GCACAATTTT 6822TTCCAGGAAA GAATCTCTCT AACTCCTTCA GCTCACATGC TTTGGACCAC ACAGGGAAGA 6882CTTTGATTGT GTAATGCCCT CAGAAGCTCT CCTTCTTGCC ACTACCACAC TGATTTGAGG 6942AAGAAAATCC CTTTAGCACC TAACCCTTCA GGTGCTATGA GTGGCTAATG GAACTGTACC 7002TCCTTCAAGT TTTGTGCAAT AATTAAGGGT CACTCACTGT CAGATACTTT CTGTGATCTA 7062TGATAATGTG TGTGCAACAC ATAACATTTC AATAAAAGTA GAAAATATGA AATTAGAGTC 7122ATCTACACAT CTGGATTTGA TCTTAGAATG AAACAAGCAA AAAAGCATCC AAGTGAGTGC 7182AATTATTAGT TTTCAGAGAT GCTTCAAAGG CTTCTAGGCC CATCCCGGGA AGTGTTAATG 7242AGCTGTGGAC TGGTTCACAT ATCTATTGCC TCTTGCCAGA TTTGCAAAAA ACTTCACTCA 7302ATGAGCAAAT TTCAGCCTTA AGAAACAAAG TCAAAAATTC CAAGGAAGCA TCCTACGAAA 7362GAGGGAACTT CTGAGATCCC TGAGGAGGGT CAGCATGTGA TGGTTGTATT TCCTTCTTCT 7422CAG T ACT GCA GAC TAT GCC ATG TTC AAG GTG GGA CCT GAA GCT GAC 7468 Thr Ala Asp Tyr Ala Met Phe Lys Val Gly Pro Glu Ala Asp 285 290 295AAG TAC CGC CTA ACA TAT GCC TAC TTC GCT GGT GGG GAT GCT GGA GAT 7516Lys Tyr Arg Leu Thr Tyr Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp 300 305 310GCC TTT GAT GGC TTT GAT TTT GGC GAT GAT CCT AGT GAC AAG TTT TTC 7564Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe315 320 325 330ACA TCC CAT AAT GGC ATG CAG TTC AGT ACC TGG GAC AAT GAC AAT GAT 7612Thr Ser His Asn Gly Met Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp 335 340 345AAG TTT GAA GGC AAC TGT GCT GAA CAG GAT GGA TCT GGT TGG TGG ATG 7660Lys Phe Glu Gly Asn Cys Ala Glu Gln Asp Gly Ser Gly Trp Trp Met 350 355 360AAC AAG TGT CAC GCT GGC CAT CTC AAT GGA GTT TAT TAC CAA G 7703Asn Lys Cys His Ala Gly His Leu Asn Gly Val Tyr Tyr Gln 365 370 375GTATGTTTTC CTTTCTTAGA TTCCAAGTTA ATGTATAGTG TATACTATTT TCATAAAAAA 7763TAATAAATAG ATATGAAGAA ATGAAGAATA ATTTATAAAG ATAGTAGGGA TTTTATCATG 7823TTCTTTATTT CAACTAAGTT CTTTGAAACT GGAAGTGGAT AATACCAAGT TCATGCCTAA 7883AATTAGCCCT TCTAAAGAAA TCCACCTGCT GCAAAATATC CAGTAGTTTG GCATTATATG 7943TGAAACTATC ACCATCATAG CTGGCACTGT GGGTTGTGGG ATCTCCTTTA GACATACAAC 8003ATAAATGATC TGGATGGATT AACATTACTA CATGGATGCT TGTTGACACA TTAACCTGGC 8063TTCCCATGAG CTTTGTGTCA GATACACGCA GTGAACAGGT GTTTGGAGGA ACAGAATAAA 8123GAGAAGGCAA GCACTGGTAA GGGCAGGGGT TTGTGAAAGC TTGAGAGAAG AGACCAGTCT 8183GAGGACAGTA GACACTTATT TTAGGATGGG GGTTGGATGA GGAGGCTATA GTTTGCTATA 8243AGCTTGGAAT GGTTTGGAAC ACTGGTTTCA CTCACCTACC CAGCAGTTAT GTGTGGGGAA 8303GCCTTACCGA TGCTAAAGGA TCCATGTTAC AATAATGGCA TTATTTGGAA ATCCCAGTGG 8363TATTCCATGA ATAAAACCAC TATGAAGATA ATCCCACTCA ACAGACTCTC CGTTGGAGAA 8423GGACAGCAAC ACCACCCTGG GAAAGCCAAA CAGTCAGACC AGACCTGTTT AGCATCAGTA 8483GGACTTCCCT ACCATATCTG CTGGGTAGAT GAGTGAAACC AGTGTTCCAA ACCACTCCGG 8543GCTTGTAGCA AACCATAGTC TCCTCATCTA CCAAGATGAG CAACCTTACC TCCTGATGTC 8603CTAGCCAATC ACCAACTAGG AAACTTTGCA CAGTTTATTT AAAGTAACAG TTTGATTTTC 8663ACAATATTTT TAAATTGGAG AAACATAACT TATCTTTGCA CTCACAAACC ACATAATGAG 8723AAGAAACTCT AAGGGAAAAT GCTTGATCTG TGTGACCCGG GGCGCCATGC CAGAGCTGTA 8783GTTCATGCCA GTGTTGTGCT CTGACAAGCC TTTTACAGAA TTACATGAGA TCTGCTTCCC 8843TAGGACAAGG AGAAGGCAAA TCAACAGAGG CTGCACTTTA AAATGGAGAC ATAAAATAAC 8903ATGCCAGAAC CATTTCCTAA AGCTCCTCAA TCAACCAACA AAATTGTGCT TTCAAATAAC 8963CTGAGTTGAC CTCATCAGGA ATTTTGTGGC TCCTTCTCTT CTAACCTGCC TGAAGAAAGA 9023TGGTCCACAG CAGCTGAGTC CGGGATGGAT AAGCTTAGGG ACAGAGGCCA ATTAGGGAAC 9083TTTGGGTTTC TAGCCCTACT AGTAGTGAAT AAATTTAAAG TGTGGATGTG ACTATGAGTC 9143ACAGCACAGA TGTTGTTTAA TAATATGTTT ATTTTATAAA TTGATATTTT AGGAATCTTT 9203GGAGATATTT TCAGTTAGCA GATAATACTA TAAATTTTAT GTAACTGGCA ATGCACTTCG 9263TAATAGACAG CTCTTCATAG ACTTGCAGAG GTAAAAAGAT TCCAGAATAA TGATATGTAC 9323ATCTACGACT TGTTTTAG GT GGC ACT TAC TCA AAA GCA TCT ACT CCT AAT 9373 Gly Gly Thr Tyr Ser Lys Ala Ser Thr Pro Asn 380 385GGT TAT GAT AAT GGC ATT ATT TGG GCC ACT TGG AAA ACC CGG TGG TAT 9421Gly Tyr Asp Asn Gly Ile Ile Trp Ala Thr Trp Lys Thr Arg Trp Tyr 390 395 400TCC ATG AAG AAA ACC ACT ATG AAG ATA ATC CCA TTC AAC AGA CTC ACA 9469Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn Arg Leu Thr 405 410 415ATT GGA GAA GGA CAG CAA CAC CAC CTG GGG GGA GCC AAA CAG GTC AGA 9517Ile Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys Gln Val Arg420 425 430 435CCA GAG CAC CCT GCG GAA ACA GAA TAT GAC TCA CTT TAC CCT GAG GAT 9565Pro Glu His Pro Ala Glu Thr Glu Tyr Asp Ser Leu Tyr Pro Glu Asp 440 445 450GAT TTG TAGAAAATTA ACTGCTAACT TCTATTGACC CACAAAGTTT CAGAAATTCT 9621Asp LeuCTGAAAGTTT CTTCCTTTTT TCTCTTACTA TATTTATTGA TTTCAAGTCT TCTATTAAGG 9681ACATTTAGCC TTCAATGGAA ATTAAAACTC ATTTAGGACT GTATTTCCAA ATTACTGATA 9741TCAGAGTTAT TTAAAAATTG TTTATTTGAG GAGATAACAT TTCAACTTTG TTCCTAAATA 9801TATAATAATA AAATGATTGA CTTTATTTGC ATTTTTATGA CCACTTGTCA TTTATTTTGT 9861CTTCGTAAAT TATTTTCATT ATATCAAATA TTTTAGTATG TACTTAATAA AATAGGAGAA 9921CATTTTAGAG TTTCAAATTC CCAGGTATTT TCCTTGTTTA TTACCCCTAA ATCATTCCTA 9981TTTAATTCTT CTTTTTAAAT GGAGAAAATT ATGTCTTTTT AATATGGTTT TTGTTTTGTT 10041ATATATTCAC AGGCTGGAGA CGTTTAAAAG ACCGTTTCAA AAGAGATTTA CTTTTTTAAA 10101GGACTTTATC TGAACAGAGA GATATAATAT TTTTCCTATT GGACAATGGA CTTGCAAAGC 10161TTCACTTCAT TTTAAGAGCA AAAGACCCCA TGTTGAAAAC TCCATAACAG TTTTATGCTG 10221ATGATAATTT ATCTACATGC ATTTCAATAA ACCTTTTGTT TCCTAAGACT AGATACATGG 10281TACCTTTATT GACCATTAAA AAACCACCAC TTTTTGCCAA TTTACCAATT ACAATTGGGC 10341AACCATCAGT AGTAATTGAG TCCTCATTTT ATGCTAAATG TTATGCCTAA CTCTTTGGGA 10401GTTACAAAGG AAATAGCAAT TATGGCTTTT GCCCTCTAGG AGATACAGGA CAAATACAGG 10461AAAATACAGC AACCCAAACT GACAATACTC TATACAAGAA CATAATCACT AAGCAGGAGT 10521CACAGCCACA CAACCAAGAT GCATAGTATC CAAAGTGCAG CTG 10564453 amino acidsamino acidlinearprotein 6Met Ser Trp Ser Leu His Pro Arg Asn Leu Ile Leu Tyr Phe Tyr Ala 1 5 10 15Leu Leu Phe Leu Ser Ser Thr Cys Val Ala Tyr Val Ala Thr Arg Asp 20 25

30Asn Cys Cys Ile Leu Asp Glu Arg Phe Gly Ser Tyr Cys Pro Thr Thr 35 40 45Cys Gly Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp Lys 50 55 60Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val Glu Asn Lys Thr 65 70 75 80Ser Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn Pro 85 90 95Asp Glu Ser Ser Lys Pro Asn Met Ile Asp Ala Ala Thr Leu Lys Ser 100 105 110Arg Ile Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile Leu Thr 115 120 125His Asp Ser Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn Ser Asn Asn 130 135 140Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln Leu Glu Ala Gln145 150 155 160Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr Gly 165 170 175Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly Leu 180 185 190Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val Tyr Cys 195 200 205Glu Ile Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg Leu 210 215 220Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln Tyr Lys Glu Gly225 230 235 240Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly Asn 245 250 255Glu Lys Ile His Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala Leu 260 265 270Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala Asp Tyr 275 280 285Ala Met Phe Lys Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr Tyr 290 295 300Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp Ala Phe Asp Gly Phe Asp305 310 315 320Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly Met 325 330 335Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn Cys 340 345 350Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His Ala Gly 355 360 365His Leu Asn Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser 370 375 380Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala Thr Trp Lys Thr385 390 395 400Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn 405 410 415Arg Leu Thr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly Ala Lys 420 425 430Gln Val Arg Pro Glu His Pro Ala Glu Thr Glu Tyr Asp Ser Leu Tyr 435 440 445Pro Glu Asp Asp Leu 45010807 base pairsnucleic aciddoublelinearovine beta-lactoglobulin 7ACGCGTGTCG ACCTGCAGGT CAACGGATCT CTGTGTCTGT TTTCATGTTA GTACCACACT 60GTTTTGGTGG CTGTAGCTTT CAGCTACAGT CTGAAGTCAT AAAGCCTGGT ACCTCCAGCT 120CTGTTCTCTC TCAAGATTGT GTTCTGCTGT TTGGGTCTTT AGTGTCTCCA CACAATTTTT 180AGAATTGTTT GTTCTAGTTC TGTGAAAAAT GATGCTGGTA TTTTGATAAG GATTGCATTG 240AATCTGTAAA GCTACAGATA TAGTCATTGG GTAGTACAGT CACTTTAACA ATATTAACTC 300TTCACATCTG TGAGCATGAT ATATTTTCCC CCTCTATATC ATCTTCAATT CCTCCTATCA 360GTTTCTTTCA TTGCAGTTTT CTGAGTACAG GTCTTACACC TCCTTGGTTA GAGTCATTCC 420TCAGTATTTT ATTCCTTTGA TACAATTGTG AATGAGGTAA TTTTCTTAGT TTCTCTTTCT 480GATAGCTCAT TGTTAGTGTA TATATAGAAA AGCAACAGAT TTCTATGTAT TAATTTTGTA 540TCCTGCAACA GATTTCTATG TATTAATTTT GTATCCTGCT ACTTTACGGA ATTCACTTAT 600TAGCTTTTTG GTGACATCTT GAGGATTTTC TGAAGAAAAT GGCATGGTAT GGTAGGACAA 660GGTGTCATGT CATCTGCAAA CAGTGGCAGT TTTCCTTCTT CCCTTCCAAC CTGGATTTCT 720TTGATTTCTT TCTGTCTGAG TACGACTAGG ATTCCCAATA CTATACCGAA TAAAAGTGGC 780AAGAGTGGAC ATCCTTGTCT TATTTTTCTG ACCTTAGAGG AAATGCTTTC AGTTTTTCAC 840CATTAATTAT AATGTTTACT GTGGGCTTGT CATATGTGGC CTTCATTATA TGGAGGTCTA 900TTCCCTCTAT ACCCACCTTG TTGAGAGTTT TTATCATAAA AGTATGTTGA ATTTTGTCAA 960AAGTTTTTCC TGCATCTATT GAGATGATTT TTACTCTTCA ATTCATTAAT GATTTTTATT 1020CTTCATTTTG TTAATGATTT CCATTCTTCA ATTTGTTAAC GTGGTATATC ACATTGATTG 1080ATTTGTGGAT ACCTTTGTAT CCCTGGGATA AACCTCACTT GATCATGAGC TTTCAATGTA 1140TTTTTGAATT CACTTTGCTA ATATTCTGTT GGGTATTTTT GCATCTCTAT TCATCAATGA 1200TATTGGCCTA AGAAAGGTTT TGTCTGGTTT TAGTATCAGG GTGATGCTGG CCTCATAGAG 1260AGAGTTTAGA AGCATTTCCT CCTCTTTGAT TTTTCGGAAT AGTTTGAGTA GGATAGGTAT 1320TAACTCTTCT TTAAATGTTT GGGGACTTCC CTGGTGAGCC GGTGGTTGAG AATCCGCCTC 1380AGGGATGTGG GTTTGATCCC TGGTCAGGGA ACCATTAATA AGATCCCACA TGCTGCAGGC 1440AACAAGCCCC CAAGCTGCAA CCACTGAGCT GCAACCGCTG CAGTGCCCAC AGGCCACGAC 1500CAGAGAAAGC CCACATACAG CAGGGAAGAC CCAGCACAAC CGGAAAAAGG AGTTTGGTGG 1560AATACAGCTG TGAAGCCGTC TGGTCCTGGA CTCCTGCTTG AGGGAATTTT TTAAAAATTA 1620TTGATTCAAT TTCATTACTG GTAACTGGTC TGTTCATATT TTCTATTTCT TCCGGGTTCA 1680GTCTTGGGAG ATTGTACATG CCTAGGAATG TGTCCGTTTC TTCTAGGTTG TCCATTTTAT 1740TGGACATGCA TGGGAGCACA CAGCACCGAC CAGCGAGACT CATGCTGGCT TCCTGGGGCC 1800AGGCTGGGGC CCCAAGCAGC ATGGCATCCT AGAGTGTGTG AAAGCCCACT GACCCTGCCC 1860AGCCCCACAA TTTCATTCTG AGAAGTGATT CCTTGCTTCT GCACTTACAG GCCCAGGATC 1920TGACCTGCTT CTGAGGAGCA GGGGTTTTGG CAGGACGGGG AGATGCTGAG AGCCGACGGG 1980GGTCCAGGTC CCCTCCCAGG CCCCCCTGTC TGGGGCAGCC CTTGGGAAAG ATTGCCCCAG 2040TCTCCCTCCT ACAGTGGTCA GTCCCAGCTG CCCCAGGCCA GAGCTGCTTT ATTTCCGTCT 2100CTCTCTCTGG ATGGTATTCT CTGGAAGCTG AAGGTTCCTG AAGTTATGAA TAGCTTTGCC 2160CTGAAGGGCA TGGTTTGTGG TCACGGTTCA CAGGAACTTG GGAGACCCTG CAGCTCAGAC 2220GTCCCGAGAT TGGTGGCACC CAGATTTCCT AAGCTCGCTG GGGAACAGGG CGCTTGTTTC 2280TCCCTGGCTG ACCTCCCTCC TCCCTGCATC ACCCAGTTCT GAAAGCAGAG CGGTGCTGGG 2340GTCACAGCCT CTCGCATCTA ACGCCGGTGT CCAAACCACC CGTGCTGGTG TTCGGGGGGC 2400TACCTATGGG GAAGGGCTTC TCACTGCAGT GGTGCCCCCC GTCCCCTCTG AGATCAGAAG 2460TCCCAGTCCG GACGTCAAAC AGGCCGAGCT CCCTCCAGAG GCTCCAGGGA GGGATCCTTG 2520CCCCCCCGCT GCTGCCTCCA GCTCCTGGTG CCGCACCCTT GAGCCTGATC TTGTAGACGC 2580CTCAGTCTAG TCTCTGCCTC CGTGTTCACA CGCCTTCTCC CCATGTCCCC TCCGTGTCCC 2640CGTTTTCTCT CACAAGGACA CCGGACATTA GATTAGCCCC TGTTCCAGCC TCACCTGAAC 2700AGCTCACATC TGTAAAGACC TAGATTCCAA ACAAGATTCC AACCTGAAGT TCCCGGTGGA 2760TGTGAGTTCT GGGGCGACAT CCTTCAACCC CATCACAGCT TGCAGTTCAT CGCAAAACAT 2820GGAACCTGGG GTTTATCGTA AAACCCAGGT TCTTCATGAA ACACTGAGCT TCGAGGCTTG 2880TTGCAAGAAT TAAAGGTGCT AATACAGATC AGGGCAAGGA CTGAAGCTGG CTAAGCCTCC 2940TCTTTCCATC ACAGGAAAGG GGGGCCTGGG GGCGGCTGGA GGTCTGCTCC CGTGAGTGAG 3000CTCTTTCCTG CTACAGTCAC CAACAGTCTC TCTGGGAAGG AAACCAGAGG CCAGAGAGCA 3060AGCCGGAGCT AGTTTAGGAG ACCCCTGAAC CTCCACCCAA GATGCTGACC AGCCAGCGGG 3120CCCCCTGGAA AGACCCTACA GTTCAGGGGG GAAGAGGGGC TGACCCGCCA GGTCCCTGCT 3180ATCAGGAGAC ATCCCCGCTA TCAGGAGATT CCCCCACCTT GCTCCCGTTC CCCTATCCCA 3240ATACGCCCAC CCCACCCCTG TGATGAGCAG TTTAGTCACT TAGAATGTCA ACTGAAGGCT 3300TTTGCATCCC CTTTGCCAGA GGCACAAGGC ACCCACAGCC TGCTGGGTAC CGACGCCCAT 3360GTGGATTCAG CCAGGAGGCC TGTCCTGCAC CCTCCCTGCT CGGGCCCCCT CTGTGCTCAG 3420CAACACACCC AGCACCAGCA TTCCCGCTGC TCCTGAGGTC TGCAGGCAGC TCGCTGTAGC 3480CTGAGCGGTG TGGAGGGAAG TGTCCTGGGA GATTTAAAAT GTGAGAGGCG GGAGGTGGGA 3540GGTTGGGCCC TGTGGGCCTG CCCATCCCAC GTGCCTGCAT TAGCCCCAGT GCTGCTCAGC 3600CGTGCCCCCG CCGCAGGGGT CAGGTCACTT TCCCGTCCTG GGGTTATTAT GACTCTTGTC 3660ATTGCCATTG CCATTTTTGC TACCCTAACT GGGCAGCAGG TGCTTGCAGA GCCCTCGATA 3720CCGACCAGGT CCTCCCTCGG AGCTCGACCT GAACCCCATG TCACCCTTGC CCCAGCCTGC 3780AGAGGGTGGG TGACTGCAGA GATCCCTTCA CCCAAGGCCA CGGTCACATG GTTTGGAGGA 3840GCTGGTGCCC AAGGCAGAGG CCACCCTCCA GGACACACCT GTCCCCAGTG CTGGCTCTGA 3900CCTGTCCTTG TCTAAGAGGC TGACCCCGGA AGTGTTCCTG GCACTGGCAG CCAGCCTGGA 3960CCCAGAGTCC AGACACCCAC CTGTGCCCCC GCTTCTGGGG TCTACCAGGA ACCGTCTAGG 4020CCCAGAGGGG ACTTCCTGCT TGGCCTTGGA TGGAAGAAGG CCTCCTATTG TCCTCGTAGA 4080GGAAGCCACC CCGGGGCCTG AGGATGAGCC AAGTGGGATT CCGGGAACCG CGTGGCTGGG 4140GGCCCAGCCC GGGCTGGCTG GCCTGCATGC CTCCTGTATA AGGCCCCAAG CCTGCTGTCT 4200CAGCCCTCCA CTCCCTGCAG AGCTCAGAAG CACGACCCCA GGGATATCCC TGCAGCCATG 4260AAGTGCCTCC TGCTTGCCCT GGGCCTGGCC CTCGCCTGTG GCGTCCAGGC CATCATCGTC 4320ACCCAGACCA TGAAAGGCCT GGACATCCAG AAGGTTCGAG GGTTGGCCGG GTGGGTGAGT 4380TGCAGGGCGG GCAGGGGAGC TGGGCCTCAG AGAGCCAAGA GAGGCTGTGA CGTTGGGTTC 4440CCATCAGTCA GCTAGGGCCA CCTGACAAAT CCCCGCTGGG GCAGCTTCAA CCAGGCGTTC 4500ACTGTCTTGC ATTCTGGAGG CTGGAAGCCC AAGATCCAGG TGTTGGCAGG GCTGGCTTCT 4560CCTGCGGCCG CTCTCTGGGG AGCAGACGGC CGTCTTCTCC AGTCCTCTGC GCGCCCTGAT 4620TTCCTCTTCC TGTGAGGCCA CCAGGCCTGC TGGAAACACG CCTGCCTGCG CAGCTTCACA 4680CGACCTTTGT CATCTCTTTA AAGGCCATGT CTCCAGAGTC ATGTGTTGAA GTTCTGGGGG 4740TTAGTGGGAC ACAGTTCAGC CCCTAAAAGA GTCTCTCTGC CCCTCAAATT TTCCCCACCT 4800CCAGCCATGT CTCCCCAAGA TCCAAATGTT GCTACATGTG GGGGGGCTCA TCTGGGTCCC 4860TCTTTGGGTT CAGTGTGAGT CTGGGGAGAG CATTCCCCAG GGTGCAGAGT TGGGGGGAGT 4920ATCTCAGGGC TGCCCAGGCC GGGGTGGGAC AGAGAGCCCA CTGTGGGGCT GGGGGCCCCT 4980TCCCACCCCC AGAGTGCAAC TCAAGGTCCC TCTCCAGGTG GCGGGGACTT GGCACTCCTT 5040GGCTATGGCG GCCAGCGACA TCTCCCTGCT GGATGCCCAG AGTGCCCCCC TGAGAGTGTA 5100CGTGGAGGAG CTGAAGCCCA CCCCCGAGGG CAACCTGGAG ATCCTGCTGC AGAAATGGTG 5160GGCGTCTCTC CCCAACATGG AACCCCCACT CCCCAGGGCT GTGGACCCCC CGGGGGGTGG 5220GGTGCAGGAG GGACCAGGGC CCCAGGGCTG GGGAAGAGGG CTCAGAGTTT ACTGGTACCC 5280GGCGCTCCAC CCAAGGCTGC CCACCCAGGG CTTTTTTTTT TTTTAAACTT TTATTAATTT 5340GATGCTTCAG AACATCATCA AACAAATGAA CATAAAACAT TCATTTTTGT TTACTTGGAA 5400GGGGAGATAA AATCCTCTGA AGTGGAAATG CATAGCAAAG ATACATACAA TGAGGCAGGT 5460ATTCTGAATT CCCTGTTAGT CTGAGGATTA CAAGTGTATT TGAGCAACAG AGAGACATTT 5520TCATCATTTC TAGTCTGAAC ACCTCAGTAT CTAAAATGAA CAAGAAGTCC TGGAAACGAA 5580GCAGTGTGGG GATAGGCCCG TGTGAAGGCT GCTGGGAGGC AGCAGACCTG GGTCTTCGGG 5640CTCAAGCAGT TCCCGCTACC AGCCCTGTCC ACCTCAGACG GGGGTCAGGG TGCAGGAGAG 5700AGCTGGATGG GTGTGGGGGC AGAGATGGGG ACCTGAACCC CAGGGCTGCC TTTTGGGGGT 5760GCCTGTGGTC AAGGCTCTCC CTGACCTTTT CTCTCTGGCT TCATCTGACT TCTCCTGGCC 5820CATCCACCCG GTCCCCTGTG GCCTGAGGTG ACAGTGAGTG CGCCGAGGCT AGTTGGCCAG 5880CTGGCTCCTA TGCCCATGCC ACCCCCCTCC AGCCCTCCTG GGCCAGCTTC TGCCCCTGGC 5940CCTCAGTTCA TCCTGATGAA AATGGTCCAT GCCAATGGCT CAGAAAGCAG CTGTCTTTCA 6000GGGAGAACGG CGAGTGTGCT CAGAAGAAGA TTATTGCAGA AAAAACCAAG ATCCCTGCGG 6060TGTTCAAGAT CGATGGTGAG TCCGGGTCCC TGGGGGACAC CCACCACCCC CGCCCCCGGG 6120GACTGTGGAC AGGTTCAGGG GGCTGGCGTC GGGCCCTGGG ATGCTAAGGG ACTGGTGGTG 6180ATGAAGACAC TGCCTTGACA CCTGCTTCAC TTGCCTCCCC TGCCACCTGC CCGGGGCCTT 6240GGGGCGGTGG CCATGGGCAG GTCCCGGCTG GCGGGCTAAC CCACCAGGGT GACACCCGAG 6300CTCTCTTTGC TGGGGGGCGG GCGGTGCTCT GGGCCCTCAG GCTGAGCTCA GGAGGTACCT 6360GTGCCCTCCC AGGGGTAACC GAGAGCCGTT GCCCACTCCA GGGGCCCAGG TGCCCCACGA 6420CCCCAGCCCG CTCCACAGCT CCTTCATCTC CTGGAGACAA ACTCTGTCCG CCCTCGCTCA 6480TTCACTTGTT CGTCCTAAAT CCGAGATGAT AAAGCTTCGA GGGGGGGTTG GGGTTCCATC 6540AGGGCTGCCC TTCCGCCGGG CAGCCTGGGC CACATCTGCC CTTGGCCCCC TCAGGACTCA 6600CTCTGACTGG AGGCCCTGCA CTGACTGACG CCAGGGTGCC CAGCCCAGGG TCTCTGGCGC 6660CATCCAGCTG CACTGGGTTT GGGTGCTGGT CCTGCCCCCA AGCTGCCCGG ACACCACAGG 6720CAGCCGGGGC TGCCCACTGG CCTCGGTCAG GGTGAGCCCC AGCTGCCCCC GCTCAGGGCT 6780TGCCCCGACA ATGACCCCAT CCTCAGGACG CACCCCCCTT CCCTTGCTGG GCAGTGTCCA 6840GCCCCACCCG AGATCGGGGG AAGCCCTATT TCTTGACAAC TCCAGTCCCT GGGGGAGGGG 6900GCCTCAGACT GAGTGGTGAG TGTTCCCAAG TCCAGGAGGT GGTGGAGGGT CCTGGCGGAT 6960CCAGAGTTGA CAGTGAGGGC TTCCTGGGCC CCATGCGCCT GGCAGTGGCA GCAGGGAAGA 7020GGAAGCACCA TTTCAGGGGT GGGGGATGCC AGAGGCGCTC CCCACCCCGT CTTCGCCGGG 7080TGGTGACCCC GGGGGAGCCC CGCTGGTCGT GGAGGGTGCT GGGGGCTGAC TAGCAACCCC 7140TCCCCCCCCG TTGGAACTCA CTTTTCTCCC GTCTTGACCG CGTCCAGCCT TGAATGAGAA 7200CAAAGTCCTT GTGCTGGACA CCGACTACAA AAAGTACCTG CTCTTCTGCA TGGAAAACAG 7260TGCTGAGCCC GAGCAAAGCC TGGCCTGCCA GTGCCTGGGT GGGTGCCAAC CCTGGCTGCC 7320CAGGGAGACC AGCTGCGTGG TCCTTGCTGC AACAGGGGGT GGGGGGTGGG AGCTTGATCC 7380CCAGGAGGAG GAGGGGTGGG GGGTCCCTGA GTCCCGCCAG GAGAGAGTGG TCGCATACCG 7440GGAGCCAGTC TGCTGTGGGC CTGTGGGTGG CTGGGGACGG GGGCCAGACA CACAGGCCGG 7500GAGACGGGTG GGCTGCAGAA CTGTGACTGG TGTGACCGTC GCGATGGGGC CGGTGGTCAC 7560TGAATCTAAC AGCCTTTGTT ACCGGGGAGT TTCAATTATT TCCCAAAATA AGAACTCAGG 7620TACAAAGCCA TCTTTCAACT ATCACATCCT GAAAACAAAT GGCAGGTGAC ATTTTCTGTG 7680CCGTAGCAGT CCCACTGGGC ATTTTCAGGG CCCCTGTGCC AGGGGGGCGC GGGCATCGGC 7740GAGTGGAGGC TCCTGGCTGT GTCAGCCGGC CCAGGGGGAG GAAGGGACCC GGACAGCCAG 7800AGGTGGGGGG CAGGCTTTCC CCCTGTGACC TGCAGACCCA CTGCACTGCC CTGGGAGGAA 7860GGGAGGGGAA CTAGGCCAAG GGGGAAGGGC AGGTGCTCTG GAGGGCAAGG GCAGACCTGC 7920AGACCACCCT GGGGAGCAGG GACTGACCCC CGTCCCTGCC CCATAGTCAG GACCCCGGAG 7980GTGGACAACG AGGCCCTGGA GAAATTCGAC AAAGCCCTCA AGGCCCTGCC CATGCACATC 8040CGGCTTGCCT TCAACCCGAC CCAGCTGGAG GGTGAGCACC CAGGCCCCGC CCTTCCCCAG 8100GGCAGGAGCC ACCCGGCCCC GGGACGACCT CCTCCCATGG TGACCCCCAG CTCCCCAGGC 8160CTCCCAGGAG GAAGGGGTGG GGTGCAGCAC CCCGTGGGGG CCCCCTCCCC ACCCCCTGCC 8220AGGCCTCTCT TCCCGAGGTG TCCAGTCCCA TCCTGACCCC CCCATGACTC TCCCTCCCCC 8280ACAGGGCAGT GCCACGTCTA GGTGAGCCCC TGCCGGTGCC TCTGGGGTAA GCTGCCTGCC 8340CTGCCCCACG TCCTGGGCAC ACACATGGGG TAGGGGGTCT TGGTGGGGCC TGGGACCCCA 8400CATCAGGCCC TGGGGTCCCC CCTGTGAGAA TGGCTGGAAG CTGGGGTCCC TCCTGGCGAC 8460TGCAGAGCTG GCTGGCCGCG TGCCACTCTT GTGGGTGACC TGTGTCCTGG CCTCACACAC 8520TGACCTCCTC CAGCTCCTTC CAGCAGAGCT AAGGCTAAGT GAGCCAGAAT GGTACCTAAG 8580GGGAGGCTAG CGGTCCTTCT CCCGAGGAGG GGCTGTCCTG GAACCACCAG CCATGGAGAG 8640GCTGGCAAGG GTCTGGCAGG TGCCCCAGGA ATCACAGGGG GGCCCCATGT CCATTTCAGG 8700GCCCGGGAGC CTTGGACTCC TCTGGGGACA GACGACGTCA CCACCGCCCC CCCCCCATCA 8760GGGGGACTAG AAGGGACCAG GACTGCAGTC ACCCTTCCTG GGACCCAGGC CCCTCCAGGC 8820CCCTCCTGGG GCTCCTGCTC TGGGCAGCTT CTCCTTCACC AATAAAGGCA TAAACCTGTG 8880CTCTCCCTTC TGAGTCTTTG CTGGACGACG GGCAGGGGGT GGAGAAGTGG TGGGGAGGGA 8940GTCTGGCTCA GAGGATGACA GCGGGGCTGG GATCCAGGGC GTCTGCATCA CAGTCTTGTG 9000ACAACTGGGG GCCCACACAC ATCACTGCGG CTCTTTGAAA CTTTCAGGAA CCAGGGAGGG 9060ACTCGGCAGA GACATCTGCC AGTTCACTTG GAGTGTTCAG TCAACACCCA AACTCGACAA 9120AGGACAGAAA GTGGAAAATG GCTGTCTCTT AGTCTAATAA ATATTGATAT GAAACTCAAG 9180TTGCTCATGG ATCAATATGC CTTTATGATC CAGCCAGCCA CTACTGTCGT ATCAACTCAT 9240GTACCCAAAC GCACTGATCT GTCTGGCTAA TGATGAGAGA TTCCCAGTAG AGAGCTGGCA 9300AGAGGTCACA GTGAGAACTG TCTGCACACA CAGCAGAGTC CACCAGTCAT CCTAAGGAGA 9360TCAGTCCTGG TGTTCATTGG AGGACTGATG TTGAAGCTGA AACTCCAATG CTTTGGCCAC 9420CTGATGTGAA GAGCTGACTC ATTTGAAAAG ACCCTGATGC TGGGAAAGAT TGAGGGCAGG 9480AGGAGAAGGG GACGACAGAG GATGAGATGG TTGGATGGCA TCACCAACAC AATGGACATG 9540GGTTTGGGTG GACTCCAGGA GTTGGTGATG GACAGGGAGG CCTGGCGTGC TACGGAAGCG 9600GTTTATGGGG TCACAAAGAC TGAGTGACTG AACTGAGCTG AACTGAATGG AAATGAGGTA 9660TACAGCAAAG TGGGGATTTT TTAGATAATA AGAATATACA CATAACATAG TGTATACTCA 9720TATTTTTATG CATACCTGAA TGCTCAGTCA CTCAGTCGTA TCTGACTCTG TGACCTATGG 9780ACCGTAGCCT TCCAGGTTTC TTCTGTCCAC AGAATTCTCC AAGGCAAGAA TACTGGAGTG 9840GGTAGCCATT TCCTCCTCCA GGGGATCCTC CCGACCCAGG GATTGAACCG GCATCTCCTG 9900TATTGGCAGG TGGATTCTTT ACCACTGTGC CACCAGGGAA GCCCGTGTTA CTCTCTATGT 9960CCCACTTAAT TACCAAAGCT GCTCCAAGAA AAAGCCCCTG TGCCCTCTGA GCTTCCCGGC 10020CTGCAGAGGG TGGTGGGGGT AGACTGTGAC CTGGGAACAC CCTCCCGCTT CAGGACTCCC 10080GGGCCACGTG ACCCACAGTC CTGCAGACAG CCGGGTAGCT CTGCTCTTCA AGGCTCATTA 10140TCTTTAAAAA AAACTGAGGT CTATTTTGTG ACTTCGCTGC CGTAACTTCT GAACATCCAG 10200TGCGATGGAC AGGACCTCCT CCCCAGGCCT CAGGGGCTTC AGGGAGCCAG CCTTCACCTA 10260TGAGTCACCA GACACTCGGG GGTGGCCCCG CCTTCAGGGT GCTCACAGTC TTCCCATCGT 10320CCTGATCAAA GAGCAAGACC AATGACTTCT TAGGAGCAAG CAGACACCCA CAGGACACTG 10380AGGTTCACCA GAGCTGAGCT GTCCTTTTGA ACCTAAAGAC ACACAGCTCT CGAAGGTTTT 10440CTCTTTAATC TGGATTTAAG GCCTACTTGC CCCTCAAGAG GGAAGACAGT CCTGCATGTC 10500CCCAGGACAG CCACTCGGTG GCATCCGAGG CCACTTAGTA TTATCTGACC GCACCCTGGA 10560ATTAATCGGT CCAAACTGGA CAAAAACCTT GGTGGGAAGT TTCATCCCAG AGGCCTCAAC 10620CATCCTGCTT TGACCACCCT GCATCTTTTT TTCTTTTATG TGTATGCATG TATATATATA 10680TATATATTTT TTTTTTTTTC ATTTTTTGGC TGTGCTGGCT GTTCGTTGCA GTTCGGTGCG 10740CAGGCTTCTC TCTAGTTTCT CTCTAGTCTT CTCTTATCAC AGAGCAGTCT CTAGACGATC 10800GACGCGT 1080747 base pairsnucleic acidsinglelinear 8AATTCCGATC GACGCGTCGA CGATATACTC TAGACGATCG ACGCGTA 4724 base pairsnucleic acidsinglelinearBLGAMP3 9TGGATCCCCT GCCGGTGCCT CTGG 2424 base pairsnucleic acidsinglelinearBLGAMP4 10AACGCGTCAT CCTCTGTGAG CCAG 2410 base pairsnucleic acidsinglelinearZC6839 11ACTACGTAGT 1042 base pairsnucleic acidsinglelinearZC6632 12CGACGCGGAT CCTACGTACC TGCAGCCATG TTTTCCATGA GG 4221 base pairsnucleic acidsinglelinearZC6627 13AGGGCTTCGG CAAGCTTCAG G 2124 base pairsnucleic acidsinglelinearZC6521 14GCCAAAGACT TACTTCCCTC TAGA 2430 base pairsnucleic acidsinglelinearZC6520 15GCATGAACGT CGCGTGGTGG TTGTGCTACC 3030 base pairsnucleic acidsinglelinearZC6519 16ACCACGCGAC GTTCATGCTC TAAAACCGTT 3036 base pairsnucleic acidsinglelinearZC6518 17GCTGCGGGAT CCTACGTACT AGGGGGACAG GGAAGG 3645 base pairsnucleic acidsinglelinearZC6629 18CGACGCGAAT TCTACGTACC TGCAGCCATG AAAAGGATGG TTTCT 4545 base pairsnucleic acidsinglelinearZC6630 19CGACGCGAAT TCTACGTACC TGCAGCCATG AAACATCTAT TATTG 4521 base pairsnucleic acidsinglelinearZC6625 20GTGAGATTTT CAGATCTTGT C 2121 base pairsnucleic acidsinglelinearZC6626 21AAGAATTACT GTGGCCTACC A 2133 base pairsnucleic acidsinglelinearZC6624 22GCTGCGGAAT TCTACGTACT ATTGCTGTGG GAA 3345 base pairsnucleic acidsinglelinearZC6514 23CGACGCGGAT CCTACGTACC TGCAGCCATG AGTTGGTCCT

TGCAC 4521 base pairsnucleic acidsinglelinearzc6517 24GTCTCTGGTA GCAACATACT A 2122 base pairsnucleic acidsinglelinearzc6516 25GGGTTTCTAG CCCTACTAGT AG 2222 base pairsnucleic acidsinglelinearzc6515 26GGGTTTCTAG CCCTACTAGT AG 2247 base pairsnucleic acidsinglelinear 27AAGCTACGCG TCGATCGTCT AGAGTATATC GTCGACGCGT CGATCGG 47

Claims

We claim:

1. A method for producing biocompetent fibrinogen comprising: providing a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen A.alpha. chain, .Iadd.the DNA segment comprising genomic DNA encoding the A.alpha. chain, .Iaddend.a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, .Iadd.the DNA segment comprising genomic DNA encoding the B.beta. chain, .Iaddend.and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. chain, .Iadd.the DNA segment comprising genomic DNA encoding the .gamma. chain, .Iaddend.wherein each chain is from the same species, and wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal .Iadd.and the first, second, third segments are linked in a single vector.Iaddend.; introducing said DNA segments into a fertilized egg of a non-human mammalian species heterologous to the species of origin of said fibrinogen chains; inserting said egg into an oviduct or uterus of a female of said mammalian species to obtain offspring carrying said DNA segments; breeding said offspring to produce female progeny that express said first, second and third DNA segments and produce milk containing biocompetent fibrinogen encoded by said segments; collecting milk from said female progeny; and and recovering the biocompetent fibrinogen from the milk.

2. A method according to claim 1 wherein said species into which said DNA segments are introduced is selected from the group consisting of sheep, pigs, goats, and cattle.

.[.3. A method according to claim 1 wherein each of said first, second and third DNA segments comprises an intron..].

.[.4. A method according to claim 1 wherein the molar ratio of said first, second and third DNA segments is within the range of 0.5-1:0.5-1:0.5-1..].

5. A method according to claim 1 wherein each of said first, second and third DNA segments is operably linked to a transcription promoter selected from the group consisting of casein, .beta.-lactoglobulin, .alpha.-lactalbumin and whey acidic protein gene promoters.

6. A method according to claim 1 wherein said first, second and third DNA segments are expressed under the control of a .beta.-lactoglobulin promoter.

7. A method according to claim 1 wherein said introducing step comprises injecting said first, second and third DNA segments into a pronucleus of said fertilized egg.

8. A method according to claim 1 wherein said fibrinogen is human fibrinogen.

9. A method according to claim 1 wherein said second DNA segment comprises a sequence of nucleotides as shown in SEQ ID NO: 3 from nucleotide 470 to nucleotide 8100.

10. A method according to claim 1 wherein said second DNA segment comprises a sequence of nucleotides as shown in SEQ ID NO: 3 from nucleotide 512 to nucleotide 8100.

11. A method according to claim 1 wherein said species into which said DNA segments is introduced is sheep.

12. A method of producing biocompetent fibrinogen comprising: incorporating .Iadd.into operable linkage .Iaddend.a .[.first.]. DNA segment encoding a secretion signal .[.operably linked to.]..Iadd., a genomic DNA segment encoding .Iaddend.an A.alpha. chain of fibrinogen .[.into a .beta.-lactoglobulin gene.]. .Iadd.and an additional segment required for expression of the A.alpha..Iaddend. chain in the mammary gland of a mammal to produce a first gene fusion.[.comprising a .beta.-lactoglobulin promoter operably linked to the first DNA segment.].; incorporating .Iadd.into operable linkage .Iaddend.a .[.second.]. DNA segment encoding a secretion signal .[.operably linked to.]..Iadd., a genomic DNA segment encoding .Iaddend.a B.beta. chain of fibrinogen .[.into a .beta.-lactoglobulin gene.]. .Iadd.and an additional segment required for expression of the B.beta. chain .Iaddend.to produce a second gene fusion .[.comprising a .beta.-lactoglobulin promoter operably linked to the second DNA segment.].; incorporating .Iadd.into operable linkage .Iaddend.a .[.third.]. DNA segment encoding a secretion signal .[.operably linked to.]..Iadd., a genomic DNA segment encoding .Iaddend.a .gamma. chain of fibrinogen .Iadd.and an additional segment required for expression of the .Iaddend..gamma. chain .[.into a .beta.-lactoglobulin gene.]. to produce a third gene fusion.Iadd., .Iaddend..[.comprising a .beta.-lactoglobulin promoter operably linked to the third DNA segment.]. wherein each of said first, second and third segments are of the same species; .Iadd.linking the first, second and third gene fusions in a single vector; .Iaddend.introducing said first, second and third gene fusions into the germ line of a non-human mammal so that said DNA segments are expressed in a mammary gland of said mammal or its female progeny and biocompetent fibrinogen is secreted into milk of said mammal or its female progeny; obtaining milk from said mammal or its female progeny; and recovering said fibrinogen from said milk.

13. A method according to claim 12 wherein said mammal is a sheep, pig, goat or cow.

.[.14. A method according to claim 12 wherein each of said first, second and third gene fusions comprises an intron..].

.[.15. A method according to claim 12 wherein the molar ratio of said first, second and third gene fusions introduced is within the range of 0.5-1:0.5-1:0.5-1..].

16. A method according to claim 12 wherein said introducing step comprises injecting said first, second and third gene fusions into a pronucleus of a fertilized egg and inserting said egg into an oviduct of a pseudopregnant female to produce female offspring carrying said gene fusions in the germ line, wherein said egg and said pseudopregnant female are of the same species.

17. A method according to claim 12 wherein said mammal is a sheep.

.[.18. A method for producing biocompetent fibrinogen comprising: providing a transgenic female non-human mammal carrying in its germline heterologous DNA segments encoding A.alpha., B.beta. and .gamma. chains of flbrinogen, wherein said segments are expressed in a mammary gland of said mammal and biocompetent fibrinogen encoded by said segments is secreted into milk of said mammal; collecting milk from said mammal; and recovering said biocompetent fibrinogen from said milk..].

.[.19. A method according to claim 18 wherein said mammal is a sheep, pig, goat or cow..].

.[.20. A method according to claim 18 wherein said mammal is a sheep..].

.[.21. A transgenic non-human female mammal that produces recoverable amounts of biocompetent human fibrinogen in its milk, wherein said mammal comprises: a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen A.alpha. chain, a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. chain, and further wherein each chain is derived from the same species and is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal..].

.[.22. A mammal according to claim 21 wherein said mammal is a sheep..].

23. A process for producing a transgenic offspring of a mammal comprising: providing a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen A.alpha. chain, .Iadd.the DNA segment comprising genomic DNA encoding the A.alpha. chain; .Iaddend.a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, .Iadd.the DNA segment comprising genomic DNA encoding the B.beta. chain; .Iaddend.and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. chain, .Iadd.the DNA segment comprising genomic DNA encoding the .gamma. chain; .Iaddend.wherein each chain is derived from the same species, and wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal; .Iadd.linking the first, second and third segments in a single vector;.Iaddend. introducing said DNA segments into a fertilized egg of a non-human mammalian species heterologous to the species of origin of said fibrinogen chains; inserting said fertilized egg into an oviduct or uterus of a female of said mammalian species; and allowing said fertilized egg to develop thereby producing transgenic offspring carrying said first, second and third DNA segments, wherein female progeny of said mammal express said DNA segments in a mammary gland to produce biocompetent fibrinogen.

24. A process according to claim 23 wherein said offspring is female.

25. A process according to claim 23 wherein said offspring is male.

.[.26. A non-human mammal produced according to the process of claim 23..].

.[.27. A non-human mammal according to claim 26 wherein said mammal is female..].

.[.28. A non-human female mammal according to claim 27 that produces milk containing biocompetent fibrinogen encoded by said DNA segments..].

.[.29. A non-human mammal according to claim 26 wherein said mammal is male..].

.[.30. A non-human mammal carrying in its germline DNA segments encoding human A.alpha., B.beta. and .gamma. chains of fibrinogen, wherein female progeny of said mammal express said DNA segments in a mammary gland to produce biocompetent human fibrinogen..].

.[.31. A mammal non-human according to claim 30 wherein said mammal is female..].

.[.32. A mammal non-human according to claim 30 wherein said mammal is male..].

.[.33. A mammal according to claim 30, wherein said mammal is a sheep..].

.Iadd.34. A set of DNA sequences comprising: a first DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen A.alpha. chain, the DNA segment comprising genomic DNA encoding the A.alpha. chain; a second DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen B.beta. chain, the DNA segment comprising genomic DNA encoding the B.beta. chain; and a third DNA segment encoding a secretion signal operably linked to a heterologous fibrinogen .gamma. chain, the DNA segment comprising genomic DNA encoding the .gamma. chain, wherein each chain is from the same species, and wherein each of said first, second and third segments is operably linked to additional DNA segments required for its expression in the mammary gland of a host female mammal; and the first, second, third segments are linked in a single vector..Iaddend.