NZ561999A - Marker assisted selection of bovine for milk fat colour - Google Patents

Abstract

Provided is a method of determining genetic merit of a bovine with respect to milk or tissue colour or beta-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or beta-carotene content, which comprises the step of determining the scavenger receptor class 1, member 1 (SCARB1) allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the SCARB1 allelic profile. Further provided are bovines selected by the method and milk obtained from such selected bovines.

Description

■ 561999 4 " NEW ZEALAND PATENTS ACT, 1953 No: 561999 Date: 26 September 2007 COMPLETE SPECIFICATION MARKER ASSISTED SELECTION OF BOVINE FOR DESIRED MILK OR TISSUE CONTENT We, VIALACTIA BIOSCIENCES (NZ) LIMITED, a company incorporated under the laws of New Zealand of 9 Princes Street, Auckland, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 561999 2 MARKER ASSISTED SELECTION OF BOVINE FOR DESIRED MILK OR TISSUE CONTENT FIELD OF THE INVENTION id="p-1" id="p-1"

[0001] This invention relates to an application of marker assisted selection of bovine for a quantitative trait loci (QTL) associated with milk and tissue colour and p-carotcnc content, particularly by assaying for the presence of polymorphisms in a gene which is associated with the QTL.

BACKGROUND id="p-2" id="p-2"

[0002] The genetic basis of bovine milk production is of immense significance to the dairy industry. An ability to modulate milk volumes and composition has the potential to alter farming practices and to produce products which are tailored to meet a range of requirements. In particular, a method of genetically evaluating bovine to select those which express desirable traits, such as desirable milk fat colour or composition, would be useful. Similarly, a method of genetically evaluating bovine with desirable tissue colour or composition would be useful. id="p-3" id="p-3"

[0003] Genetic bases for variations in the composition of milk, for example, the relative amounts of major milk proteins, and the effect of these variations on milk production characteristics and milk processing properties, has been the subject of considerable research, debate, and review. For example, PCT International application PCT/NZ01/00245 (published as W002/36824) reports that polymorphisms in the bovine Diacylglycerol-o-acyltransferase (DGAT1) gene are associated with increased milk yield and altered milk composition, and in particular that the presence of a K232A mutation in the DGAT1 gene results in a decrease in milk fat percentage, milk fat yield, solid fat content and milk protein percentage, while increasing milk volume and milk protein yield. In another example, PCT International application PCT/NZ02/00157 (published as W003/104492) reports that polymorphisms in the bovine growth hormone receptor (GHR) gene are associated with an increased milk volume and altered milk composition, and in particular that the presence of the F279Y amino acid variant results in increased milk yield and decreased milk fat and milk protein percentage, as well as a decrease in live weight. For other characteristics of milk composition, the basis for variation is less clear. id="p-4" id="p-4"

[0004] The yellow colour of milk and milk fat, caused primarily by the presence of (J-carotene, is considered a negative characteristic in some consumer markets. Conversely, 561999 3 other markets prize the yellow colour, while foods enriched in p-carotene have been associated with health benefits. Consequently, strategies to modulate milk colour could be economically valuable. Although environmental factors, such as diet, lactation stage and milk volume, influence milk colour, previous research suggests that some of the variation in milk colour may be attributable to genetics (Winkelman et al., 1999). Similarly, different consumer markets variously perceive tissue colour. id="p-5" id="p-5"

[0005] Strategies to modulate milk colour or (3-carotcne content or tissue colour or p-carotene content could provide health benefits and are expected to be economically valuable. (3-carotene and vitamin A deficiencies are still major health problems (particularly in developing countries) leading to blindness and childhood mortality. Milk with increased P-carotene content would be of benefit, for example in markets where other dietary sources of p-carotene are scarce or not commonly consumed. id="p-6" id="p-6"

[0006] Marker assisted selection, which provides the ability to follow a specific favourable genetic allele, involves the identification of a DNA molecular marker or markers that segregate(s) with a gene or group of genes associated with or which in part defines a trait. DNA markers have several advantages. They are relatively easy to measure and are unambiguous, and as DNA markers are co-dominant, heterozygous and homozygous animals can be distinctively identified. Once a marker system is established, selection decisions are able to be made very easily as DNA markers can be assayed at any time after a DNA containing sample has been collected from an individual animal, whether embryonic, infant or adult. id="p-7" id="p-7"

[0007] It is an object of the present invention to provide a method for marker assisted selection of bovine with desired milk colour or milk P-carotene content or tissue colour or tissue p-carotene content; and/or to provide animals selected using the method of the invention as well as milk produced by or tissue(s) derived from the selected animals; and/or to provide the public with a useful choice.

SUMMARY OF THE INVENTION id="p-8" id="p-8"

[0008] This invention relates to the elucidation of the role of the gene encoding Scavenger receptor class B, member 1 (SCARB1, also known as CD36 antigen, collagen type I receptor, and thrombospondin receptor-like 1) in milk or tissue colour or p-carotene content, particularly milk fat colour and p-carotene content. In particular, the invention relates to the identification of the C-321G promoter polymorphism in the SCARB1 gene, and to the 561999 4 association of the C allele with production of milk and particularly milk fat with increased |i-carotene content for the first time. id="p-9" id="p-9"

[0009] This gives rise to numerous, and separate, aspects of the invention. id="p-10" id="p-10"

[0010] In one aspect the invention provides a method of determining the genetic merit of a bovine with respect to milk or tissue colour or p-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or p-carotene content, which comprises determining the SCARB1 allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the SCARB1 allelic profile. id="p-11" id="p-11"

[0011] In one embodiment, milk p-carotene content is milk fat p-carotene content. id="p-12" id="p-12"

[0012] In another embodiment, milk content is milk colour, more preferably milk fat colour. id="p-13" id="p-13"

[0013] In still further embodiments, the tissue is selected from liver or adipose tissue. id="p-14" id="p-14"

[0014] In one embodiment, the genetic merit with respect to milk or tissue colour or p-carotene content is production of milk or tissue with increased colour or P-carotene content. id="p-15" id="p-15"

[0015] Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk, tissue, milk fat, or tissue fat with increased P-carotene content, or that is capable of producing progeny that produce milk, tissue, milk fat, or tissue fat with increased P-carotene content, comprising determining the expression or activity of the SCARB1 gene product, and identifying or selecting the bovine on the basis of the determination. id="p-16" id="p-16"

[0016] In another embodiment, the genetic merit with respect to milk or tissue colour or p-carotene content is production of milk or tissue with decreased colour or p-carotene content. Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk, tissue, milk fat, or tissue fat with decreased p-carotene content, or capable of producing progeny that produce milk, tissue, milk fat, or tissue fat with decreased P-carotene content, comprising determining the expression or activity of the SCARE 1 gene product, and identifying or selecting the bovine on the basis of the determination. id="p-17" id="p-17"

[0017] In one embodiment, the method comprises determining the expression or activity of a SCARB1 gene or gene product, and preferably identifying or selecting the bovine on the basis of the determination. id="p-18" id="p-18"

[0018] In one embodiment, expression or activity of the SCARB1 gene product is determined using SCARB1 mRNA, for example by determining the presence or amount of 561999 SCARBl mRNA. In other embodiments, expression or activity of the SCARBl gene product Is determined using SCARBl protein, preferably by determining the amount of SCARBl protein, for example the amount of SCARBl protein, or by determining the activity of SCARBl protein, for example the enzymatic activity of SCARBl protein present in a sample obtained from the bovine. It will be apparent that the activity of SCARBl protein (such as that present in a sample obtained from the bovine) may be determined by determining the localisation of SCARBl or of SCARBl ligand, for example by determining the ratio of intracellular SCARBl to extracellular SCARBl, or by determining the the ratio of intracellular SCARBl ligand to extracellular SCARBl ligand, or by determining the transport or uptake of SCARBl or of SCARBl ligand across, out of, or into a cell or membrane. In still other embodiments, the expression or activity of the SCARBl gene product is determined using SCARBl DNA, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased SCARBl expression or activity, for example one or more promoter polymorphisms associated with increased or decreased expression, or one or more coding sequence polymorphisms associated with increased or decreased expression or activity. id="p-19" id="p-19"

[0019] In another embodiment, the SCARB1 allelic profile of the bovine is determined together with the allelic profile of the bovine at one or more genetic loci associated with milk or tissue colour or p-carotene content. id="p-20" id="p-20"

[0020] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or tissue colour or j3-carotene content. id="p-21" id="p-21"

[0021] The one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with said one or more polymorphisms. id="p-22" id="p-22"

[0022] Linkage disequilibrium (LD) is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co-inherited. This means that in genotyping, detection of one polymorphism as present infers the presence of the other. (Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 411:199-204.) id="p-23" id="p-23"

[0023] It will be apparent that as used herein, the phrase "SCARBl allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the SCARBl gene or which affect expression from the SCARBl gene or the expression or activity of a SCARBl gene product or which are associated with variation 561999 6 in the expression from the SCARBl gene or in the expression or activity of a SCARBl gene product. In preferred embodiments, the SCARBl allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms associated with increased or decreased milk or tissue colour or P-carotene content. For example, in preferred embodiments the SCARBl allelic profile comprises data indicative of the presence or absence of the C allele, or of the presence or absence of the G allele, at the C-321G promoter polymorphism in the SCARBl gene. In other embodiments, the SCARBl allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the promoter of the SCARBl gene, or in a regulatory region of the SCARBl gene, or in an intron of the SCARBl gene, or in a coding region of the SCARBl gene, and preferably comprises data indicative of the presence or absence of one or more alleles which affect expression from the SCARBl gene or the expression or activity of a SCARBl gene product or which are associated with variation in the expression from the SCARBl gene or in the expression or activity of a SCARBl gene product. id="p-24" id="p-24"

[0024] It will further be appreciated that the SCARBl allelic profile may comprise information correlating the presence or absence of one or more polymorphisms as described above with milk or tissue colour or P-carotene content. id="p-25" id="p-25"

[0025] In one embodiment, the allelic profile is determined using nucleic acid obtained from said bovine, preferably DNA obtained from said bovine, or alternatively, said allelic profile is determined using RNA obtained from said bovine. id="p-26" id="p-26"

[0026] In yet a further embodiment, the allelic profile is determined with reference to the amino acid sequence of expressed SCARBl protein obtained from said bovine. id="p-27" id="p-27"

[0027] In another embodiment, the allelic profile is determined with reference to the amount or activity of SCARBl protein obtained from said bovine. id="p-28" id="p-28"

[0028] Conveniently, in said method the presence or absence of DNA encoding a reference SCARBl gene product, or of nucleotide sequence comprising a reference SCARBl gene, in said bovine is determined, directly or indirectly, for example using an expressed SCARBl gene product. id="p-29" id="p-29"

[0029] Alternatively, in said method the presence or absence of at least one nucleotide difference from the nucleotide sequence of a reference SCARBl gene, for example, at least one nucleotide difference from the nucleotide sequence encoding reference SCARBl, in said bovine is determined, directly or indirectly. 561999 7 id="p-30" id="p-30"

[0030] In one embodiment, the method comprises determining the SCARBl C-321G promoter allelic profile of the bovine. id="p-31" id="p-31"

[0031] More specifically, in said method the presence or absence of one or more of the C allele or G allele at the C-321G promoter polymorphism in the SCARBl gene is determined, directly or indirectly. For example, the presence of the C allele or of the G allele at the C-321G promoter polymorphism in the SCARBl gene may be determined using a polymorphism in linkage disequilibrium with the C allele or with the G allele at the C-321G promoter polymorphism. id="p-32" id="p-32"

[0032] In one embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a sequence of the DNA encoding a protein "(A)" having biological activity of reference SCARBl is present, or whether a sequence of the DNA encoding an allelic protein "(B)" at least partially lacking the activity of (A) is present, or whether a sequence of the DNA encoding (A) and a sequence of the DNA encoding (B) are both present. id="p-33" id="p-33"

[0033] As used herein, biological activity of reference SCARBl protein refers to both expression levels and activity characteristic of SCARBl protein expressed from the reference SCARBl gene. id="p-34" id="p-34"

[0034] In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a reference SCARBl gene sequence is present. In still another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, the expression of the SCARBl gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased SCARBl expression, for example one or more promoter polymorphisms associated with increased or decreased expression. id="p-35" id="p-35"

[0035] In one embodiment, this method includes ascertaining whether a sequence of the mRNA encoding a protein "(A)" having biological activity of a reference SCARBl is present, or whether a sequence of the mRNA encoding a protein "(B)" at least partially lacking the activity of (A) is present, or whether a sequence of the mRNA encoding (A) and a sequence of the mRNA encoding (B) are both present. id="p-36" id="p-36"

[0036] In another embodiment, the method includes ascertaining the amount of SCARBl mRNA present in a sample of material containing mRNA obtained from the bovine. 561999 8 id="p-37" id="p-37"

[0037] In another embodiment, the method includes ascertaining whether a protein "(A)" having biological activity of a reference SCARBl is present, or whether a protein "(B)" at least partially lacking the activity of (A) is present, or whether (A) and (B) are both present. id="p-38" id="p-38"

[0038] In another embodiment, the method includes ascertaining the amount or activity of SCARBl protein present in a sample of material containing protein obtained from the bovine. id="p-39" id="p-39"

[0039] In another aspect, the invention is a method for determining the SCARBl genotype of a bovine, as may be desirable to know for breeding purposes. id="p-40" id="p-40"

[0040] In one embodiment, the method includes ascertaining, with reference to a sample of material containing nucleic acid obtained from the bovine and uncontaminated by heterologous nucleic acid, whether the sample contains (i) nucleic acid molecule encoding a protein having biological activity of reference SCARBl and optionally ascertaining whether the sample contains an (ii) allelic nucleic acid molecule encoding a protein lacking biological activity of reference SCARBl. id="p-41" id="p-41"

[0041] In another embodiment, the method includes ascertaining, with reference to a sample of material containing protein obtained from the bovine and uncontaminated by heterologous protein, whether the sample contains (i) a protein having biological activity of reference SCARBl and optionally ascertaining whether the sample contains a protein lacking biological activity of reference SCARB1. id="p-42" id="p-42"

[0042] In a further embodiment, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue |3-carotene content which comprises determining the SCARBl allelic profile of the bovine, together with determining the allelic profile of the bovine at one or more genetic loci associated with milk or tissue p-carotene content. id="p-43" id="p-43"

[0043] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or tissue p-carotene content, preferably one or more polymorphisms in one or more genes involved in P-carotene uptake or metabolism. id="p-44" id="p-44"

[0044] In a further aspect, the invention includes a probe comprising a nucleic acid molecule sufficiently complementary with a nucleic acid sequence comprising a bovine SCARBl gene or encoding a bovine SCARBl gene product, or its complement, so as to bind thereto under stringent conditions, as well as a diagnostic kit containing such a probe. ]0045] The invention also includes a primer composition useful for detection of the presence or absence of a reference SCARBl gene and/or the presence or absence of nucleic 561999 9 acid encoding reference SCARB1. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising a reference SCARBl gene or encoding reference SCARBl, or its complement. The nucleic acid sequence can in whole or in part be identified in SEQ ID No.l or SEQ ID No.3. The invention also includes a primer composition useful for detection of the presence or absence of a variant SCARBl gene and/or the presence of the DNA encoding a variant SCARBl protein at least partially lacking reference SCARBl activity. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising a variant SCARBl gene or encoding a variant SCARBl protein, or its complement. The nucleic acid sequence can in whole or in part be identified in SEQ ID NO:l or SEQ ID NO:3. Diagnostic kits including such a composition are also included. id="p-46" id="p-46"

[0046] Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in SEQ ID NO:l and within approximately 1 to about 2000 bp of the C-321G promoter polymorphism, more preferably within approximately 1 to about 1000 bp, or within approximately 1 to about 500 bp, approximately I to about 400 bp, approximately 1 to about 300 bp, approximately 1 to about 200 bp, approximately 1 to about 100 bp, approximately I to about 50 bp, or approximately 1 to about 20 bp of the C-321G promoter polymorphism. id="p-47" id="p-47"

[0047] It will be appreciated by those skilled in the art that a pair of such primers can be used to determine the identity of the nucleotide at the C-321G promoter polymorphism, by, for example the selective generation of an amplicon with one or more sequence-specific primers. Primer compositions comprising a pair of such primers are accordingly contemplated. id="p-48" id="p-48"

[0048] The invention also provides a diagnostic kit including a primer composition useful for determining the presence or absence of a reference SCARBl gene and/or the presence or absence of nucleic acid encoding reference SCARBl, the diagnostic kit comprising one or more primers or primer compositions as described herein. id="p-49" id="p-49"

[0049] The invention further includes an antibody composition useful for determining the presence or absence of reference SCARBl protein, or for determining the presence or absence of a variant protein at least partially lacking reference SCARBl activity, or for determining the expression of SCARBl protein, as well as a diagnostic kit containing such an antibody together with instructions for use, for example in a method of the invention. 561999 id="p-50" id="p-50"

[0050] The invention further provides a diagnostic kit useful in detecting DNA comprising a variant SCARBl gene, or DNA or mRNA encoding a variant SCARBl gene at least partially lacking reference activity, in a bovine which includes first and second primers for amplifying the DNA or mRNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a polymorphism in the SCARBl gene which results in or is associated with increased or decreased milk or tissue colour or [3-carotene content. id="p-51" id="p-51"

[0051] In one embodiment at least one of the nucleotide sequences is selected to be from a non-coding region of the reference SCARBl gene. id="p-52" id="p-52"

[0052] The kit can also include a primer complementary to a naturally occurring mutation of a coding or non-coding portion of the reference SCARBl gene, for example a mutation in the promoter of the SCARBl gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention. id="p-53" id="p-53"

[0053] Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue p-carotene content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the C-321G promoter polymorphism in the SCARBl gene, or one or more polymorphisms in linkage disequilibrium with the C-321G promoter polymorphism in the SCARBl gene. id="p-54" id="p-54"

[0054] In another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue colour or j3-carotene content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the C allele at the C-321G promoter polymorphism, or the G allele at the C-321 G promoter polymorphism. id="p-55" id="p-55"

[0055] Again, the one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with the one or more polymorphisms. id="p-56" id="p-56"

[0056] Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue p-carotene content which comprises the step of determining the presence or absence of the G allele at the C-321 G promoter polymorphism in the SCARBl gene. The invention further provides a method of assessing 561999 11 the genetic merit of a bovine with respect to milk or tissue p-carotenc content which comprises the step of determining the presence or absence of the C allele at the C-321 G promoter polymorphism in the SCARBl gene. id="p-57" id="p-57"

[0057] In another aspect, the present invention provides a method for selecting a bovine with a genotype indicative of desired milk colour or of desired milk p-carotene content or of desired tissue colour or of desired tissue p-carotene content. The method comprises determining the bovine SCARB1 allelic profile of said bovine, and selecting the bovine on the basis of the determination. id="p-58" id="p-58"

[0058] In one embodiment, the invention provides a method for identifying or selecting a bovine with increased milk colour or increased milk p-carotene content, preferably increased milk fat colour or increased milk fat p-carotene content. id="p-59" id="p-59"

[0059] In another embodiment, the invention provides a method for identifying or selecting a bovine with increased tissue colour or increased tissue p-carotene content, preferably increased tissue fat colour or increased tissue fat P-carotene content. id="p-60" id="p-60"

[0060] In one embodiment, the invention provides a method for identifying or selecting a bovine with a SCARBl allelic profile indicative of increased milk colour or increased milk J3-carotene content, preferably of increased milk fat colour or increased milk fat (3-carotene content, id="p-61" id="p-61"

[0061] In another embodiment, the invention provides a method for identifying or selecting a bovine with a SCARBl allelic profile indicative of increased tissue colour or increased tissue P-carotene content. id="p-62" id="p-62"

[0062] Preferably the method comprises determining the presence of the C allele at the C-321 G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of the G allele at the C-321 G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. id="p-63" id="p-63"

[0063] Preferably, the method comprises determining the presence of the CC genotype at the C-321 G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. id="p-64" id="p-64"

[0064] In another embodiment, the invention provides a method for identifying or selecting a bovine with decreased milk colour or decreased milk p-carotene content, preferably decreased milk fat colour or decreased milk fat p-carotene content. 561999 12 id="p-65" id="p-65"

[0065] In another embodiment, the invention provides a method for identifying or selecting a bovine with decreased tissue colour or decreased tissue P-carotene content, preferably decreased tissue fat colour or decreased tissue fat p-carotene content. id="p-66" id="p-66"

[0066] In a further embodiment the invention provides a method for selecting a bovine with a SCARBl allelic profile indicative of decreased milk colour or decreased milk [3-carotene content, preferably of decreased milk fat colour or decreased milk fat P-carotene content. id="p-67" id="p-67"

[0067] In another embodiment, the invention provides a method for selecting a bovine with a SCARBl allelic profile indicative of decreased tissue colour or decreased tissue p-carotene content. id="p-68" id="p-68"

[0068] Preferably the method comprises determining the absence of the C allele at the C-321G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the presence of the G allele at the C-321 G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. id="p-69" id="p-69"

[0069] Preferably, the method comprises determining the presence of the GG genotype at the C-321 G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. id="p-70" id="p-70"

[0070] In one embodiment, the invention provides a method for identifying or selecting a bovine with intermediate milk colour or intermediate milk p-carotene content, preferably intermediate milk fat colour or intermediate milk fat P-carotene content, id="p-71" id="p-71"

[0071] In another embodiment, the invention provides a method for identifying or selecting a bovine with intermediate tissue colour or intermediate tissue P-carotene content, preferably intermediate tissue fat colour or intermediate tissue fat p-carotene content. id="p-72" id="p-72"

[0072] In a further embodiment the invention provides a method for selecting a bovine with a SCARB1 allelic profile indicative of intermediate milk colour or intermediate milk p-carotene content, preferably of intermediate milk fat colour or intermediate milk fat p-carotene content. id="p-73" id="p-73"

[0073] In another embodiment, the invention provides a method for selecting a bovine with a SCARBl allelic profile indicative of intermediate tissue colour or intermediate tissue P-carotene content. 561999 13 id="p-74" id="p-74"

[0074] Preferably, the method comprises determining the presence of the CG genotype at the C-321 G promoter polymorphism in the SCARBl gene, and selecting the bovine on the basis of the determination. id="p-75" id="p-75"

[0075] In one embodiment, the presence of the C allele or of the G allele is determined with respect to a SCARB1 polynucleotide (genomic DNA, mRNA or cDNA produced from mRNA) obtained from the bovine. id="p-76" id="p-76"

[0076] In one embodiment, the presence of the C allele or of the G allele is determined by sequencing a SCARBl polynucleotide obtained from the bovine. id="p-77" id="p-77"

[0077] In a further embodiment the determination comprises the step of amplifying a SCARBl polynucleotide sequence from genomic DNA, mRNA or cDNA produced from mRNA derived from said bovine, for example by PCR. id="p-78" id="p-78"

[0078] Preferably the determination is by use of primers which comprise a nucleotide sequence having at least about 12 contiguous bases of or complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO:3 or a naturally occurring flanking sequence, id="p-79" id="p-79"

[0079] In one embodiment at least one of the primers comprises sequence corresponding to at least one of the allele-specific nucleotides described herein. id="p-80" id="p-80"

[0080] In an alternative embodiment, the method comprises restriction enzyme digestion of a nucleotide derived from the bovine. Such digestion may also be performed on a product of the PCR amplification described above. id="p-81" id="p-81"

[0081] In a further embodiment, the presence of the C allele or of the G allele is determined by mass spectrometric analysis of a SCARBl polynucleotide, such as that obtained from the bovine or from a method as described herein. id="p-82" id="p-82"

[0082] In an alternative embodiment, the presence of the C allele or of the G allele is determined by hybridisation of a probe or probes comprising a nucleotide sequence of or complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO:3. id="p-83" id="p-83"

[0083] Preferably the probe or probes comprises 12 or more contiguous nucleotides of or complementary to the sequence of SEQ ID NO:l or SEQ ID NO:3. id="p-84" id="p-84"

[0084] Preferably the probe or probes comprise sequence corresponding to the C allele-specific or the G allele-specific nucleotides described herein or complements thereof. id="p-85" id="p-85"

[0085] In an alternative embodiment, the presence of the C allele or of the G allele is determined by analysis of a SCARBl polypeptide obtained from the bovine. id="p-86" id="p-86"

[0086] In a lurther aspect the invention provides a bovine selected by a process of the invention; milk produced by the selected bovine or the progeny thereof as well as dairy 561999 14 products produced from such milk; and ova or semen produced by or tissue from the selected bovine. id="p-87" id="p-87"

[0087] In still a further aspect the invention provides a method of selecting a herd of bovine, comprising selecting individuals by a method of the present invention, and segregating and collecting the selected individuals to form the herd. The invention further provides a herd of bovine so selected, as well as a herd comprising bovine produced by bovine selected by the methods described herein. id="p-88" id="p-88"

[0088] In a still further aspect, the invention provides a method of determining genetic merit of a bovine with respect to one or more milk or tissue colour or [3-carotene content traits, or with respect to capability of producing progeny predisposed to or with one or more milk or tissue colour or P-carotene content traits, the method comprising providing data about the SCARBl allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the data. id="p-89" id="p-89"

[0089] Preferably, the data about the SCARBl allelic profile comprises data representative of the presence or absence of the C allele or the G allele at the C-321 G promoter polymorphism in the SCARBl gene. id="p-90" id="p-90"

[0090] Preferably, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more milk or tissue colour or p-carotene content traits, wherein the data is representative of the genetic merit of the bovine. id="p-91" id="p-91"

[0091] Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a SCARBl gene product. id="p-92" id="p-92"

[0092] Preferably the genetic loci is the SCARBl gene (Including all regulatory elements such as the promoter, introns and 3'UTR). id="p-93" id="p-93"

[0093] In one embodiment, the one or more milk or tissue colour or p-carotene content traits Is selected from the group comprising production of or capability of producing milk with increased milk colour, production of or capability of producing milk with increased milk p-carotene content, increased tissue colour, or Increased tissue p-carotene content. id="p-94" id="p-94"

[0094] In another embodiment, the one or more milk or tissue colour or P-carotene content traits is selected from the group comprising production of or capability of producing milk with decreased milk colour, production of or capability of producing milk with decreased milk p-carotene content, decreased tissue colour, or decreased tissue p-carotene content. 561999 id="p-95" id="p-95"

[0095] Accordingly, in one embodiment the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, the method comprising providing data about the SCARBl allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the data. id="p-96" id="p-96"

[0096] Preferably, the data about the SCARBl allelic profile comprises data representative of the presence or absence of the C allele or the G allele at the C-321 G promoter polymorphism in the SCARBl gene. id="p-97" id="p-97"

[0097] Preferably, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more milk or tissue colour or p-carotene content traits, wherein the data is representative of the genetic merit of the bovine. id="p-98" id="p-98"

[0098] Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a SCARBl gene product. id="p-99" id="p-99"

[0099] Preferably the genetic loci is the SCARBl gene (including all regulatory elements such as the promoter, introns and 3'UTR). id="p-100" id="p-100"

[00100] In a further aspect the invention provides a method for identifying or selecting a bovine with respect to one or more milk or tissue colour or p-carotene content traits, the method comprising providing the result of one or more genetic tests of a sample from the bovine, and analysing the result for the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of SCARBl gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms associated with increased or decreased expression or activity of SC ARB 1 gene product, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a bovine with one or more desired milk or tissue colour or p-carotene content traits; and identifying or selecting the bovine on the basis of the result. id="p-101" id="p-101"

[00101] Preferably, the one or more polymorphisms associated with increased or decreased expression or activity of SCARBl gene product is one or more polymorphisms in the SCARBl gene. 561999 16 [00102J In a further aspect the invention provides a method for selecting a bovine with one or more desired milk or tissue colour or P-carotene content traits, the method comprising a) providing the result of one or more genetic tests of a sample from the bovine, and b) analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising: the C-321 G promoter polymorphism in the SCARBl gene, or one or more polymorphisms in linkage disequilibrium with the C-321 G promoter polymorphism in the SCARBl gene, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a bovine with one or more desired milk or tissue colour or p-carotene content traits. id="p-103" id="p-103"

[00103] In other aspects, the invention provides a system for performing one or more of the methods of the invention, said system comprising: computer processor means for receiving, processing and communicating data; storage means for storing data including a reference genetic database of the results of genetic analysis of a bovine with respect to one or more milk or tissue colour or p-carotene content traits and optionally a reference milk or tissue colour or p-carotene content trait database of non-genetic factors for one or more bovine milk or tissue colour or p-carotene content traits; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic merit of the bovine, said outcome being communicable once known, preferably to a user having input said data. id="p-104" id="p-104"

[00104] Preferably, said system is accessible via the internet or by personal computer. id="p-105" id="p-105"

[00105] Preferably, said reference genetic database comprises or Includes the results of one or more analyses of one or more genetic loci associated with one or more milk or tissue colour or p-carotene content traits, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or ]3-carotene content traits. id="p-106" id="p-106"

[00106] In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data 561999 17 consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more milk or tissue colour or (3-carotene content traits in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with one or more bovine milk or tissue colour or p-carotene content traits. id="p-107" id="p-107"

[00107] Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or p-carotene content traits. id="p-108" id="p-108"

[00108] Preferably the gene is the SCARBl gene (including all regulatory elements such as the promoter, introns and 3'UTR). id="p-109" id="p-109"

[00109] Preferably, the one or more polymorphisms are one or more polymorphisms associated with an increase or decrease in expression or activity of a SCARBl gene product. id="p-110" id="p-110"

[00110] In still another aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, the method comprising determining milk or tissue colour or p-carotene content of the bovine, determining the SCARB1 allelic profile of the bovine, comparing the SCARBl allelic profile of the bovine or the milk or tissue colour or [3-carotene content of the bovine with that of a bovine having a known SCARBl allelic profile; determining the genetic merit of the bovine on the basis of the comparison. id="p-111" id="p-111"

[00111] It will be appreciated that for the purposes of the comparison, the milk or tissue colour or P-carotene content associated with the known SCARBl allelic profile is known. It will further be appreciated that the association of milk or tissue colour or p-carotene content with a particular SCARBl allelic profile may be established by the methods described herein. id="p-112" id="p-112"

[00112] In another aspect, the invention relates to an isolated, purified or recombinant nucleic acid molecule comprising nucleotide sequence selected from the group comprising: (a) at least 12 contiguous nucleotides of SEQ ID NO:l and comprising the C-321G promoter polymorphism; or (b) any one or more of SEQ ID NOs:5 - 44; or (c) a complement of (a) or (b); or (d) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (c) under stringent conditions. 561999 18 id="p-113" id="p-113"

[00113] In one embodiment, the SCARBl nucleic acid molecule is a SCARBl fragment as defined herein, wherein the SCARBl fragment comprises the C-321G promoter polymorphism. id="p-114" id="p-114"

[00114] The invention also provides a genetic construct comprising a SCARBl nucleic acid molecule of the invention, a vector comprising the genetic construct or a nucleic acid sequence as described above, a host cell comprising the genetic construct or vector, a polypeptide encoded by a SCARBl nucleic acid molecule of the invention, an antibody which selectively binds a polypeptide of the invention, and a method for recombinantly producing a polypeptide of the invention. id="p-115" id="p-115"

[00115] The term "comprising" as used in this specification means "consisting at least in part of'. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. id="p-116" id="p-116"

[00116] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows a graph showing the range and seasonal effect of (3-carotene concentration determined as described in the Example. Data for peak (35 days post calving), mid (late November) and late (late February) lactation is shown. Figure 2 shows a graph depicting a QTL for milk fat colour on bovine chromosome 17. The maximum F-value of 10.6 occurred at 71 centimorgans (cM). The approximate map location of the SCARBl gene was between two microsatellite markers BMS1879 at 50.7 cM and BM1862 at 67.5 cM.

Figure 3 is a schematic showing a graphical representation of the C-321G promoter polymorphism in bovine SCARBl. A: Graphical representation of the predicted gene structure of bovine SCARBl where vertical bars represent exons, as well as 5' UTR and 3' UTR. B. Identification of the C/G polymorphism at position -321 relative to the +1 translation start site 561999 19 (promoter). The polymorphism (highlighted) was heterozygous in five F1 sires, and one sire was homozygous for the G allele. is a graph showing the effect of SCARBl genotype on milk fat colour. Data shown are means ± s.e.m. Data shown are for peak lactation but are representative of the additional two time points that were measured. The main effect of SCARBl genotype was significant at P < 0.01. is a graph showing the adjusted statistical effect of SCARBl genotype on the bovine chromosome 17 milk fat colour QTL. Solid line: milk fat colour QTL (residual of modeled data excluding SCARBl genotype). Dotted line: milk fat colour QTL after SCARBl genotype included in statistical model (residual of modeled data including SCARBl genotype as a fixed effect). The decrease in QTL significance (i;-value) suggests a significant association of the SCARBl genotype with the milk fat colour QTL variation. is a graph showing the p-carotene content of bovine adipose tissue. The main effect of the SCARBl genotype was significant at p < 0.01. Data shown are means ± s.e.m. Significance between specific genotype groups is indicated by asterix (* P < 0.01). For the CC, CG and GG genotype groups there were n=5, n=12 and n=7 animals, respectively. ;is a graph showing the effect SCARBl genotype on liver SCARBl mRNA levels as measured by quantitative real time PCR. Data shown are the fold change means relative to a reference sample ± s.e.m. Significance between genotype groups is indicated by asterix (* P < 0.01). For the CC, CG and GG genotype groups there were n=7, n=13 and n=8 animals, respectively. DETAILED DESCRIPTION OF THE INVENTION id="p-117" id="p-117"

[00117] The present invention recognises for the first time that a polymorphism in the SCARBl gene in bovine is associated with a QTL for variations in milk colour and milk p-carotene content, and for variations in tissue colour and tissue p-carotene content, id="p-118" id="p-118"

[00118] For the sake of clarity, the phrase "milk or tissue colour or p-carotene content" is to be read as referring to milk colour or milk p-carotene content or tissue colour or tissue p-carotene content. Grammatical equivalents or components thereof are to be read likewise, such that the phrase "milk or tissue colour" is to be read as "milk colour or tissue colour". id="p-119" id="p-119"

[00119] It will be apparent to those skilled in the art that milk or tissue colour can readily be determined qualitatively or quantitatively. For example, a visual comparison may in many Figure 4 Figure 5 Figure 6 Figure 7 561999 cases be sufficient to qualitatively determine a sample of milk or tissue having increased colour, or decreased colour, relative to another sample. Methods for quantitative determination of milk or tissue colour or p-carotene content are also known in the art, and examples are provided herein. id="p-120" id="p-120"

[00120] The invention provides methods of assessing the genetic merit of a bovine with respect to milk p-carotene content, more particularly milk fat p-carotene content. One such method comprises the step of determining the SCARBl allelic profile of said bovine. Another such method comprises the step of determining the level of the SCARBl gene product of said bovine. id="p-121" id="p-121"

[00121] The invention also provides a method for selecting a bovine with a genotype indicative of desired milk p-carotene content, particularly desired milk fat P-caiotene content. One of the major applications of the present invention is in the selection of bovine having the C allele or the G allele of the C-321 G promoter polymorphism in the SCARBl gene, which are associated with increased milk fat P-carotene content and milk fat colour, and decreased milk fat p-carotene content and milk fat colour, respectively. Accordingly, one method comprises determining the presence or absence of the C allele or of the G allele at the C-321 G promoter polymorphism of the SCARBl gene, and selecting the bovine on the basis of the determination, id="p-122" id="p-122"

[00122] Additionally, the invention is directed towards the selected bovine and semen from the selected bovine which may be useful in further breeding programs. Bovine so selected will be useful for milk production. The invention is also directed towards milk produced by the selected bovine or the progeny thereof, as well as dairy products produced from such milk. id="p-123" id="p-123"

[00123] The production of a wide variety of dairy products is well known in the art, and dairy products contemplated herein include ice creams, yoghurts and cheeses, dairy based drinks (such as milk drinks including milk shakes, and yogurt drinks), milk powders, dairy based sports supplements, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets. id="p-124" id="p-124"

[00124] Similarly, the production of a wide range of tissue products, such as but not limited to meat, organs, pelts, fluids and the like, is well known in the art. Particularly contemplated herein are meat, organs, blood and serum having increased or decreased colour or p-carotene content. 561999 21 id="p-125" id="p-125"

[00125] The present invention recognises that polymorphisms in the gene encoding SCARBl, as well as SCARBl levels or activity, may be used as a selection tool to breed animals with higher or lower milk concentrations of p-carotene (and thus milk fat colour). This in turn may allow the production of milk products more suitable to markets favouring white milk and milk products, or the production of milk products more suitable to markets favouring yellow milk and milk products, or the production of milk and milk products, such as foods, high in p-carotene. 1 SCARBl id="p-126" id="p-126"

[00126] SCARBl is believed to be involved in the uptake of carotenoids into cells. A Drosophila knockout model is reported to be blind, with no carotenoids present, and vitamin A deficient (Kiefer C., et al., 2002). id="p-127" id="p-127"

[00127] The present invention relates to the identification that a polymorphism in the SCARBl gene leads to variation in milk or tissue colour or p-carotene content, particularly variation in milk fat colour and milk fat p-carotene content, and tissue fat colour and tissue fat P-carotene content. id="p-128" id="p-128"

[00128] The genomic sequence comprising the bovine SCARBl gene is presented herein as SEQ ID NO:l. The predicted exon structure is shown in SEQ ID NO:l, and the derived amino acid sequence is presented herein as SEQ ID NO:2. The predicted coding sequence of bovine SCARBl is presented as SEQ ID NO:3, and a provisional sequence is available as NCBI accession number NM_174597.2 (GI:31341575). This coding sequence is derived from a cDNA clone, the sequence of which is available as NCBI accession number AF019384 (GI:2429347). The amino acid sequence encoded by the reference coding sequence of SEQ ID NO:3 is presented herein as SEQ ID NO:4, and is itself available as NCBI accession number NP_777022.1 (GI:27807079). id="p-129" id="p-129"

[00129] As described herein, the SCARBl C-321 G polymorphism was closely associated with milk colour phenotype. Animals homozygous for the C allele (CC genotype) produced milk with more P-carotene than heterozygous animals (CG genotype), who in turn produce milk with more p-carotene than animals homozygous for the G allele (GG genotype). This effect was observed at three stages of lactation. id="p-130" id="p-130"

[00130] The CC genotype for SCARBl was present in 23.52% of animals in the Holstein-Friesian x Jersey crossbred trial, while the CG genotype was present in 48.85% of animals, and the remaining 27.64% of animals were of GG genotype. 561999 22 id="p-131" id="p-131"

[00131] A reference bovine SCARBl nucleotide sequence is presented as SEQ ID NO:l, and the compiled reference coding sequence is presented as SEQ ID NO:3, with the corresponding amino acid sequences presented as SEQ ID NO:2, and SEQ ID NO:4, respectively. Accordingly, as used herein with respect to SCARBl, such as use with respect to a SCARBl gene or a SCARBl gene product, the term "reference" recognizes the characteristics of the SCARBl nucleotide sequences presented as SEQ ID NOs:l and 3, and of the protein product encoded thereby. For example, when used with respect to activity, the term "reference" denotes activity associated with the reference SCARBl protein. Similarly, when used with respect to expression amount, the term "reference" denotes a level of expression associated with the reference SCARBl gene or promoter. id="p-132" id="p-132"

[00132] It will be apparent that the term"activity" may refer both to the inherent activity of a single molecule of SCARBl, which may be reference activity or may be less or greater than reference activity as may depend, for example on the amino acid sequence, the presence of any amino acid substitutions, the availability of co-factors, and the like, as well as to the total activity of the population of SCARBl molecules present (for example, in a bovine or in a sample taken from a bovine), as may depend on both the activity of each molecule present and the level of expression (for example, how many such molecules are present). id="p-133" id="p-133"

[00133] As used herein, such as when used in reference to an allelic protein lacking the activity of reference SCARBl, the phrase "lacking the activity of (A)" contemplates activity both greater than that of (A) and less than that of (A). For example, an allelic protein lacking the activity of reference SCARBl may be a variant SCARBl protein of greater or lesser activity than that of reference SCARBl. id="p-134" id="p-134"

[00134] Methods to assay the activity of SCARBl are well known in the art. For example, one such method utilises quantification of SCARBl mRNA, such as that described herein. Another exemplary method utilises an assay of the uptake of p-carotene as described in During and Harrison (2007). id="p-135" id="p-135"

[00135] The genetic polymorphism identified in the bovine SCARBl gene is identified as a variant in SEQ ID NO:l and 3, and is reported in Figure 3. The nucleic acid and proteins sequences of the SCARBl G and C alleles at the C-321 G promoter polymorphic site are shown in Figure 3B. 2 Identification and analysis of polymorphisms id="p-136" id="p-136"

[00136] It will be apparent to those skilled in the field that the convention of identifying promoter polymorphisms by their position relative to the +1 translation start site of the gene 561999 23 in which they occur is followed herein. Accordingly, the C-321 G polymorphism in the SCARBl gene described herein lies 321 nucleotides upstream of the +1 translation start site of the SCARBl gene. id="p-137" id="p-137"

[00137] The C-321G promoter polymorphism can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with the C-321G promoter polymorphism. Linkage disequilibrium is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co-inherited. This means that in genotyping, detection of one polymorphism as present implies the presence of the other. (Reich DE et al; Linkage disequilibrium in the human genome, "Nature 2001, 411:199-204.) id="p-138" id="p-138"

[00138] Various degrees of linkage disequilibrium are possible. Preferably, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein are in greater than about 60% linkage disequilibrium, are in about 70% linkage disequilibrium, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% linkage disequilibrium with the C-321G promoter polymorphism of the SCARBl gene. (Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322). id="p-139" id="p-139"

[00139] There are numerous standard methods known in the art for determining whether a particular DNA sequence is present in a sample, many of which include the step of sequencing a DNA sample. Thus in one embodiment of the invention, the step determining whether or not the specified nucleotides are present in a nucleic acid derived from a bovine, includes the step of sequencing the nucleic acid. Methods for nucleotide sequencing are well known to those skilled in the art. id="p-140" id="p-140"

[00140] In one aspect, the present invention provides a method for determining the genetic merit of a bovine with respect to milk or tissue p-carotene content, and particularly with milk fat colour or p-carotene content. In one embodiment the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a sequence of the DNA encoding "(A)" a protein having biological activity of reference SCARBl is present, and whether a sequence of the DNA encoding "(B)" an allelic protein lacking the activity of (A) is present. In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether the reference SCARBl gene sequence is present. In still another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, the expression of the SCARBl 561999 24 gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased SCARBl expression, for example one or more promoter polymorphisms associated with increased or decreased expression. id="p-141" id="p-141"

[00141] An example of another art standard method known for determining whether a particular DNA sequence is present in a sample is the Polymerase Chain Reaction (PCR). One embodiment of the invention thus includes a step in which ascertaining whether a sequence of the DNA encoding (A) is present, or whether a sequence of the DNA encoding (B) is present, includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having biological activity of reference SCARBl, and/or in the presence of a primer containing at least a portion of a polymorphism known to naturally occur and which when present results in high relative p-carotene levels, and particularly in milk or tissue having inter alia a higher p-carotene content, and/or in the presence of a primer containing at least a portion of a polymorphism known to naturally occur and which when present results in low relative P-carotene levels, and particularly in milk or tissue having inter alia a lower P-carotene content. id="p-142" id="p-142"

[00142] A primer of the present invention, used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding protein of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Likewise, a probe of the present invention, is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection in the presence of nucleic acid molecules having differing sequences. id="p-143" id="p-143"

[00143] Accordingly, a preferred embodiment of the invention thus includes the step of amplifying a SCARBl polynucleotide in the presence of at least one primer comprising a nucleotide sequence of, or complementary to, the SCARBl gene (SEQ ID NO:l and SEQ ID NO:3) or flanking sequence thereof, and/or in the presence of a such a primer comprising sequence corresponding to or flanking the C-321G C allele-specific or C-321 G G allele-specific nucleotides described herein. PCR methods are well known by those skilled in the art (Mullis et al., 1994.) The template for amplification may be selected from genomic DNA, mRNA or first strand cDNA derived from a sample obtained from the bovine under test (Sambrook et al., 1989). 561999 id="p-144" id="p-144"

[00144] Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the SCARBl gene sequence, such as SEQ ID NO:l or SEQ ID NO:3 or flanking sequence thereof, and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Such primers should comprise at least about 12 contiguous bases of or complementary to SEQ ID NO:l or SEQ ID NO:3, or naturally occurring flanking sequences thereof. Examples of such PCR primers are presented herein as SEQ ID NOS:5-44. id="p-145" id="p-145"

[00145] Suitable PCR primers may include sequence corresponding to the C-321G C allele-specific or C-321G G allele-specific SCARBl nucleotides described herein. Generation of a corresponding PCR product, or the lack of product, may constitute a test for the presence or absence of the specified nucleotides in the SCARBl gene of the test bovine. id="p-146" id="p-146"

[00146] Other methods for determining whether a particular nucleotide sequence is present in a sample may include the step of restriction enzyme digestion of nucleotide sample. Separation and visualisation of the digested restriction fragments by methods well known in the art, may form a diagnostic test for the presence of a particular nucleotide sequence. The nucleotide sequence digested may be a PCR product amplified as described above. id="p-147" id="p-147"

[00147] Still other methods for determining whether a particular nucleotide sequence is present in a sample include a step of hybridisation of a probe to a sample nucleotide sequence. Thus, methods for detecting the C-321G C allele-specific or C-321G G allele-specific nucleotides may comprise the additional steps of hybridisation of a probe derived from the SCARBl sequence of SEQ ID NO:l or SEQ ID NO:3. id="p-148" id="p-148"

[00148] Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the SCARBl gene sequence, to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection of the presence or absence of the allele-specific nucleotides described herein. id="p-149" id="p-149"

[00149] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10s C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., 1989; Ausubel et al., 1987). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formulaTm = 81.5 + 0. 41% (G + C-log (Na+). id="p-150" id="p-150"

[00150] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm, On average, the Tm of 561999 26 a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)0 C. id="p-151" id="p-151"

[00151] Such a probe may be hybridised with genomic DNA, mRNA, or cDNA produced form mRNA, derived from a sample taken from a bovine under test. id="p-152" id="p-152"

[00152] Such probes would typically comprise at least 12 contiguous nucleotides of or complementary to the sequences presented SEQ ID NO:l or SEQ ID NO:3, and may comprise sequence corresponding to the allele-specific nucleotides described herein. id="p-153" id="p-153"

[00153] Such probes may additionally comprise means for detecting the presence of the probe when bound to sample nucleotide sequence. Methods for labelling probes such as radiolabelling are well known in the art (see for example, Sambrook et al., 1989). id="p-154" id="p-154"

[00154] As will be apparent to a person skilled in the art, promoter function may be determined by various well-known methods, for example use in reporter systems. For example, one approach of determining the allelic state of a promoter would be via a reporter construct, where the promoter of interest is fused upstream of a reporter gene (e.g. luciferase), and the activity of the reporter is determined and correlates with promoter function. id="p-155" id="p-155"

[00155] Accordingly, in one embodiment the method for determining the genetic merit of a bovine with respect to milk or tissue colour or (J-carotene content includes ascertaining, from a sample of material containing DNA obtained from the subject, whether a sequence of the DNA encoding the promoter of the SCARBl gene is associated with higher or lower relative levels of expression. id="p-156" id="p-156"

[00156] In another aspect, the invention provides a method for determining the genetic merit of bovine with respect to milk or tissue colour or p-carotene content with reference to a sample of material containing mRNA obtained from the bovine. In one embodiment this method includes ascertaining whether a sequence of the mRNA encoding (A) a protein having biological activity of a reference SCARBl is present, and whether a sequence of the mRNA encoding (B) a protein at least partially lacking the activity of (A) is present, and may include determining the amount of mRNA. id="p-157" id="p-157"

[00157] Again, if an amplification method such as PCR is used in ascertaining whether a sequence of the mRNA encoding (B) is present, and whether a sequence of the mRNA encoding (A) is present, the method includes amplifying the mRNA, for example in the presence of a pair of primers complementary to a nucleotide sequence encoding a protein having biological activity of a reference SCARBl, or in the presence of a pair of primers complementary to a nucleotide sequence encoding a variant SCARBl protein. It will be 561999 27 appreciated that in embodiments of the invention reliant on assessing the amount of SCARBl mRNA present in a sample, quantitative amplification methods well known in the art may be employed, for example quantitative RT-PCR, microarray analysis, and other methods described herein. id="p-158" id="p-158"

[00158] Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of mRNA are well known in the art. These include Northern analysis using probes able to hybridise to the target SCARBl mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the SCARBl coding sequence to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection and assessment of the amount of SCARBl mRNA present. As is evident to the person skilled in the art, such quantitative methods generally utilise an internal control, for example in the case of Northern analysis quantitation may be done with reference to, for example, rRNA present in the sample. id="p-159" id="p-159"

[00159] In a further aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue p-carotene content which comprises determining the SCARBl allelic profile of said bovine, together with determining the allelic profile of said bovine at one or more genetic loci associated with milk or tissue P-carotene content. id="p-160" id="p-160"

[00160] In one embodiment, said genetic loci is a polymorphism in a gene associated with milk or tissue P-carotene content, preferably a polymorphism in a gene involved in p-carotene uptake or metabolism. id="p-161" id="p-161"

[00161] The methods of the invention are reliant on genetic information such as that derived from methods suitable to the detection and identification of polymorphisms, particularly single nucleotide polymorphisms (SNPs) associated with the qualitative trait for which an assessment is desired. For the sake of convenience the following discussion refers particularly to SNPs, yet the art-skilled worker will appreciate that the methods discussed are amenable to the detection and identification of other genetic polymorphisms, such as triplet repeats or microsatellites. id="p-162" id="p-162"

[00162] A SNP is a single base change or point mutation resulting in genetic variation between individuals. SNPs are believed to occur in mammalian genomes approximately once every 100 to 300 bases, and can occur in coding or non-coding regions. Due to the redundancy of the genetic code, a SNP in the coding region may or may not change the amino acid sequence of a protein product. A SNP in a non-coding region can, for example, alter gene expression by, for example, modifying control regions such as promoters, transcription factor 561999 28 binding sites, processing sites, ribosomal binding sites, mRNA stability, and affect gene transcription, processing, and translation. id="p-163" id="p-163"

[00163] SNPs can facilitate large-scale association genetics studies, and there has recently been great interest in SNP discovery and detection. SNPs show great promise as markers for a number of phenotypic traits (including latent traits), such as for example, disease propensity and severity, wellness propensity, drug responsiveness including, for example, susceptibility to adverse drug reactions, and as described herein association with desirable phenotypic traits. Knowledge of the association of a particular SNP with a phenotypic trait, coupled with the knowledge of whether a subject has said particular SNP, can enable the targeting of diagnostic, preventative and therapeutic applications to allow better disease management, to enhance understanding of disease states, to develop selective breeding regimes, and to identify subjects of desirable genetic merit. id="p-164" id="p-164"

[00164] Indeed, a number of databases have been constructed of known SNPs, and for some such SNPs, the biological effect associated with a SNP. Understandably, there has been a focus on human genetics. For example, the NCBI SNP database "dbSNP" is incorporated into NCBl's Entrez system and can be queried using the same approach as the other Entrez databases such as PubMed and GenBank. This database has records for over 1.5 million SNPs mapped onto the human genome sequence. Each dbSNP entry includes the sequence context of the polymorphism (i.e., the surrounding sequence), the occurrence frequency of the polymorphism (by population or individual), and the experimental method(s), protocols, and conditions used to assay the variation, and can include information associating a SNP with a particular phenotypic trait. Similar databases are available for a number of species of commercial and scientific interest. id="p-165" id="p-165"

[00165] There has been and continues to be a great deal of effort to develop methods that reliably and rapidly identify new SNPs associated with a phenotypic trait. This is no trivial task, at least in part because of the complexity of mammalian genomic DNA (e.g., the haploid human genome of 3 x 109 base pairs, while current estimates of the size of the haploid bovine genome are in the range of 2.6 - 2.7 x 109 base pairs), and the associated sensitivity and discriminatory requirements. id="p-166" id="p-166"

[00166] Genotyping approaches to detect SNPs well-known in the art include DNA sequencing, methods that require allele specific hybridization of primers or probes, allele specific incorporation of nucleotides to primers bound close to or adjacent to the polymorphisms (often referred to as "single base extension", or "minisequencing"), allele- 561999 29 specific ligation (joining) of oligonucleotides (ligation chain reaction or ligation padlock probes), allele-specific cleavage of oligonucleotides or PCR products by restriction enzymes (restriction fragment length polymorphisms analysis or RFLP) or chemical or other agents, resolution of allele-dependent differences in electrophoretic or chromatographic mobilities, by structure specific enzymes including invasive structure specific enzymes, or mass spectrometry. Analysis of amino acid variation is also possible where the SNP lies in a coding region and results in an amino acid change. id="p-167" id="p-167"

[00167] DNA sequencing allows the direct determination and identification of SNPs. The benefits in specificity and accuracy are generally outweighed for screening purposes by the difficulties inherent in whole genome, or even targeted subgenome, sequencing. id="p-168" id="p-168"

[00168] Mini-sequencing involves allowing a primer to hybridize to the DNA sequence adjacent to the SNP site on the test sample under investigation. The primer is extended by one nucleotide using all four differentially tagged fluorescent dideoxynucleotides (A,C,G, or T), and a DNA polymerase. Only one of the four nucleotides (homozygous case) or two of the four nucleotides (heterozygous case) is incorporated. The base that is incorporated is complementary to the nucleotide at the SNP position. id="p-169" id="p-169"

[00169] A number of sequencing methods and platforms are particularly suited to large-scale implementation, and are amenable to use in the methods of the invention. These include pyrosequencing methods, such as that utilised in the GS FLX pyrosequencing platform available from 454 Life Sciences (Branford, CT) which can generate 100 million nucleotide data in a 7.5 hour run with a single machine, and solid-state sequencing methods, such as that utilised in the SOLiD sequencing platform (Applied Biosystems, Foster City, CA). id="p-170" id="p-170"

[00170] A number of methods currently used for SNP detection involve site-specific and/or allele-specific hybridisation. These methods are largely reliant on the discriminatory binding of oligonucleotides to target sequences containing the SNP of interest. The techniques of Illumina (San Diego, CA), Affymctrix (Santa Clara, CA.) and Nanogen Inc. (San Diego, Calif.) are particularly well-known, and utilize the fact that DNA duplexes containing single base mismatches are much less stable than duplexes that are perfectly base-paired. The presence of a matched duplex is usually detected by fluorescence. A number of whole-genome genotyping products and solutions amenable or adaptable for use in the present invention are now available, including those available from the above companies. id="p-171" id="p-171"

[00171] The majority of methods to detect or identify SNPs by site-specific hybridisation require target amplification by methods such as PCR to increase sensitivity and specificity 561999 (see, for example U.S. Pat. No. 5,679,524, PCT publication WO 98/59066, PCT publication WO 95/12607). US Application 20050059030 (incorporated herein in its entirety) describes a method for detecting a single nucleotide polymorphism in total human DNA without prior amplification or complexity reduction to selectively enrich for the target sequence, and without the aid of any enzymatic reaction. The method utilises a single-step hybridization involving two hybridization events: hybridization of a first portion of the target sequence to a capture probe, and hybridization of a second portion of said target sequence to a detection probe. Both hybridization events happen in the same reaction, and the order in which hybridisation occurs is not critical. id="p-172" id="p-172"

[00172] US Application 20050042608 (incorporated herein in its entirety) describes a modification of the method of electrochemical detection of nucleic acid hybridization of Thorp et al. (U.S. Pat. No. 5,871,918). Briefly, capture probes are designed, each of which has a different SNP base and a sequence of probe bases on each side of the SNP base. The probe bases are complementary to the corresponding target sequence adjacent to the SNP site. Each capture probe is immobilized on a different electrode having a non-conductive outer layer on a conductive working surface of a substrate. The extent of hybridization between each capture probe and the nucleic acid target is detected by detecting the oxidation-reduction reaction at each electrode, utilizing a transition metal complex. These differences in the oxidation rates at the different electrodes are used to determine whether the selected nucleic acid target has a single nucleotide polymorphism at the selected SNP site. id="p-173" id="p-173"

[00173] The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPE™ technology can genotype very large numbers of SNPs simultaneously from small or large pools of genomic material. This technology uses fluorescently labeled probes and compares the collected genomes of two populations, enabling detection and recovery of DNA fragments spanning SNPs that distinguish the two populations, without requiring prior SNP mapping or knowledge. id="p-174" id="p-174"

[00174] A number of other methods for detecting and identifying SNPs exist. These include the use of mass spectrometry, for example, to measure probes that hybridize to the SNP. This technique varies in how rapidly it can be performed, from a few samples per day to a high throughput of many thousands of SNPs per day, using mass code tags. A preferred example is the use of mass spectrometric determination of a nucleic acid sequence which comprises the polymorphisms of the invention, for example, which includes the C-321 G promoter polymorphism in the SCARBl gene (whether the coding sequence or a 561999 31 complementary sequence). Such mass spectrometric methods are known to those skilled in the art, and the genotyping methods of the invention are amenable to adaptation for the mass spectrometric detection of the polymorphisms of the invention. id="p-175" id="p-175"

[00175] SNPs can also be determined by ligation-bit analysis. This analysis requires two primers that hybridize to a target with a one nucleotide gap between the primers. Each of the four nucleotides is added to a separate reaction mixture containing DNA polymerase, ligase, target DNA and the primers. The polymerase adds a nucleotide to the 3'end of the first primer that is complementary to the SNP, and the ligase then ligates the two adjacent primers together. Upon heating of the sample, if ligation has occurred, the now larger primer will remain hybridized and a signal, for example, fluorescence, can be detected. A further discussion of these methods can be found in U.S. Pat. Nos. 5,919,626; 5,945,283; 5,242,794; and 5,952,174. id="p-176" id="p-176"

[00176] US Patent 6,821,733 (incorporated herein in its entirety) describes methods to detect differences in the sequence of two nucleic acid molecules that includes the steps of: contacting two nucleic acids under conditions that allow the formation of a four-way complex and branch migration; contacting the four-way complex with a tracer molecule and a detection molecule under conditions in which the detection molecule is capable of binding the tracer molecule or the four-way complex; and determining binding of the tracer molecule to the detection molecule before and after exposure to the four-way complex. Competition of the four-way complex with the tracer molecule for binding to the detection molecule indicates a difference between the two nucleic acids. id="p-177" id="p-177"

[00177] Protein- and proteomics-based approaches are also suitable for polymorphism detection and analysis. Polymorphisms which result in or are associated with variation in expressed proteins can be detected directly by analysing said proteins. This typically requires separation of the various proteins within a sample, by, for example, gel electrophoresis or HPLC, and identification of said proteins or peptides derived therefrom, for example by NMR or protein sequencing such as chemical sequencing or more prevalently mass spectrometry. Proteomic methodologies are well known in the art, and have great potential for automation. For example, integrated systems, such as the ProteomlQ™ system from Proteome Systems, provide high throughput platforms for proteome analysis combining sample preparation, protein separation, image acquisition and analysis, protein processing, mass spectrometry and bioinformatics technologies. 561999 32 id="p-178" id="p-178"

[00178] The majority of proteomic methods of protein identification utilise mass spectrometry, including ion trap mass spectrometry, liquid chromatography (LC) and LC/MSn mass spectrometry, gas chromatography (GC) mass spectroscopy, Fourier transform-ion cyclotron resonance-mass spectrometer (FT-MS), MALDI-TOF mass spectrometry, and ESI mass spectrometry, and their derivatives. Mass spectrometric methods are also useful in the determination of post-translational modification of proteins, such as phosphorylation or glycosyiation, and thus have utility in determining polymorphisms that result in or are associated with variation in post-translational modifications of proteins. id="p-179" id="p-179"

[00179] Associated technologies are also well known, and include, for example, protein processing devices such as the "Chemical Inkjet Printer" comprising piezoelectric printing technology that allows in situ enzymatic or chemical digestion of protein samples electroblotted from 2-D PAGE gels to membranes by jetting the enzyme or chemical directly onto the selected protein spots. After in-situ digestion and incubation of the proteins, the membrane can be placed directly into the mass spectrometer for peptide analysis. id="p-180" id="p-180"

[00180] It will be apparent that the presence or absence of the C allele or of the G allele at the C-321 G promoter polymorphism in the SCARBl gene may also be determined by analysis of a polypeptide sample, derived from a bovine. id="p-181" id="p-181"

[00181] Suitable polypeptide-based analyses include those able to discriminate between full-length and truncated protein products, and may include but are not limited to, the following: Native poly aery lamide gel electrophoresis (PAGE), isoelectric focussing, 2D PAGE, or Western blotting with specific antibodies. Mass spectroscopy, immunoprecipitation, and peptide fingerprinting are also suitable. id="p-182" id="p-182"

[00182] A large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs. id="p-183" id="p-183"

[00183] For example, Single Strand Conformational Polymorphism (SSCP, Orita et al., PNAS 1989 86:2766-2770) is a method reliant on the ability of single-stranded nucleic acids to form secondary structure in solution under certain conditions. The secondary structure depends on the base composition and can be altered by a single nucleotide substitution, causing differences in electrophoretic mobility under nondenaturing conditions. The various polymorphs are typically detected by autoradiography when radioactively labelled, by silver staining of bands, by hybridisation with detectably labelled probe fragments or the use of fluorescent PCR primers which are subsequently detected, for example by an automated DNA sequencer. 561999 33 id="p-184" id="p-184"

[00184] Modifications of SSCP are well known in the art, and include the use of differing gel running conditions, such as for example differing temperature, or the addition of additives, and different gel matrices. Other variations on SSCP are well known to the skilled artisan, including,RNA-SSCP, restriction endonuclease fmgerprinting-SSCP, dideoxy fingerprinting (a hybrid between dideoxy sequencing and SSCP), bi-directional dideoxy fingerprinting (in which the dideoxy termination reaction is performed simultaneously with two opposing primers), and Fluorescent PCR-SSCP (in which PCR products are internally labelled with multiple fluorescent dyes, may be digested with restriction enzymes, followed by SSCP, and analysed on an automated DNA sequencer able to detect the fluorescent dyes). id="p-185" id="p-185"

[00185] Other methods which utilise the varying mobility of different nucleic acid structures include Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), and Heteroduplex Analysis (HET). Here, variation in the dissociation of double stranded DNA (for example, due to base-pair mismatches) results in a change in electrophoretic mobility. These mobility shifts are used to detect nucleotide variations. id="p-186" id="p-186"

[00186] Denaturing High Pressure Liquid Chromatography (HPLC) is yet a further method utilised to detect SNPs, using HPLC methods well-known in the art as an alternative to the separation methods described above (such as gel electophoresis) to detect, for example, homoduplexes and heteroduplexes which elute from the HPLC column at different rates, thereby enabling detection of mismatch nucleotides and thus SNPs. id="p-187" id="p-187"

[00187] Yet further methods to detect SNPs rely on the differing susceptibility of single stranded and double stranded nucleic acids to cleavage by various agents, including chemical cleavage agents and nucleolytic enzymes. For example, cleavage of mismatches within RNA:DNA heteroduplexes by RNase A, of heteroduplexes by, for example bacteriophage T4 endonuclease Yll or T7 endonuclease I, of the 5' end of the hairpin loops at the junction between single stranded and double stranded DNA by cleavase I, and the modification of mispaired nucleotides within heteroduplexes by chemical agents commonly used in Maxam-Gilbert sequencing chemistry, are all well known in the art. id="p-188" id="p-188"

[00188] Further examples include the Protein Translation Test (PTT), used to resolve stop codons generated by variations which lead to a premature termination of translation and to protein products of reduced size, and the use of mismatch binding proteins. Variations are detected by binding of, for example, the MutS protein, a component of Escherichia coli DNA mismatch repair system, or the human hMSH2 and GTBP proteins, to double stranded DNA 561999 34 heteroduplexes containing mismatched bases. DNA duplexes are then incubated with the mismatch binding protein, and variations are detected by mobility shift assay. For example, a simple assay is based on the fact that the binding of the mismatch binding protein to the heteroduplex protects the heteroduplex from exonuclease degradation. id="p-189" id="p-189"

[00189] Those skilled in the art will know that a particular SNP, particularly when it occurs in a regulatory region of a gene such as a promoter, can be associated with altered expression of a gene. Altered expression of a gene can also result when the SNP is located in the coding region of a protein-encoding gene, for example where the SNP is associated with codons of varying usage and thus with tRNAs of differing abundance. Such altered expression can be determined by methods well known in the art, and can thereby be employed to detect such SNPs. Similarly, where a SNP occurs in the coding region of a gene and results in a non-synonomous amino acid substitution, such substitution can result in a change in the function of the gene product. Similarly, in cases where the gene product is an RNA, such SNPs can result in a change of function in the RNA gene product. Any such change in function, for example as assessed in an activity or functionality assay, can be employed to detect such SNPs. id="p-190" id="p-190"

[00190] The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention. 3 Polynucleotide and polypeptide variants id="p-191" id="p-191"

[00191] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. A number of nucleic acid analogues are well known in the art and are also contemplated. id="p-192" id="p-192"

[00192] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is preferably at least 15 nucleotides in length. The fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 contiguous nucleotides of a polynucleotide of the invention, A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or 561999 ribozyme technology, or as a primer, a probe, included in a microarray, or used In polynuelootide-based selection methods. id="p-193" id="p-193"

[00193] The term "fragment" in relation to promoter polynucleotide sequences is intended to include sequences comprising cis-elements and regions of the promoter polynucleotide sequence capable of regulating expression of a polynucleotide sequence to which the fragment is operably linked. id="p-194" id="p-194"

[00194] Preferably fragments of polynucleotide sequences of the invention comprise at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800, more preferably at least 900 and most preferably at least 1000 contiguous nucleotides of a polynucleotide of the invention. id="p-195" id="p-195"

[00195] The term "primer" refers to a short polynucleotide, usually having a free 3'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the template. Such a primer is preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length. id="p-196" id="p-196"

[00196] The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length. id="p-197" id="p-197"

[00197] The term "variant" as used herein refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polynucleotides and polypeptides possess biological 561999 36 activities that are the same or similar to those of the reference polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein. 3.1 Polynucleotide variants id="p-198" id="p-198"

[00198] Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, at least 100 nucleotide positions, or over the entire length of the specified polynucleotide sequence. id="p-199" id="p-199"

[00199] Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off. id="p-200" id="p-200"

[00200] The identity of polynucleotide sequences may be examined using the following unix command line parameters: id="p-201" id="p-201"

[00201] bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn 100202] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities = ". id="p-203" id="p-203"

[00203] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443- 561999 37 453). A full Implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/. id="p-204" id="p-204"

[00204] Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235. id="p-205" id="p-205"

[00205] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). id="p-206" id="p-206"

[00206] The similarity of polynucleotide sequences may be examined using the following unix command line parameters: id="p-207" id="p-207"

[00207] bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx id="p-208" id="p-208"

[00208] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match. id="p-209" id="p-209"

[00209] Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10"!0, more preferably less than 1 x 10"20, less than 1 x 10'30, less than 1 x 10"40, less than 1 x 10"50, less than 1 x 10"60, less than 1 x 10~7C, less than 1 x 10"so, less than 1 x 10~90, less than 1 x 10"i0°, less than 1 x 10~no, less than 1 x 10~120 or less than 1 x 10~123 when compared with any one of the specifically identified sequences. 561999 38 id="p-210" id="p-210"

[00210] Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions. id="p-211" id="p-211"

[00211] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency. id="p-212" id="p-212"

[00212] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30°C (for example, 10°C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C. id="p-213" id="p-213"

[00213] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10°C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)°C. id="p-214" id="p-214"

[00214] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10°C below the Tm. 561999 39 id="p-215" id="p-215"

[00215] Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism. id="p-216" id="p-216"

[00216] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306). id="p-217" id="p-217"

[00217] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bI2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described. id="p-218" id="p-218"

[00218] Polypeptide Variants id="p-219" id="p-219"

[00219] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%o, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, at least 100 amino acid positions, or over the entire length of a polypeptide of the invention. id="p-220" id="p-220"

[00220] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using 561999 40 BLASTP (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off. id="p-221" id="p-221"

[00221] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. id="p-222" id="p-222"

[00222] Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq i peptideseq 1 -j peptideseq2 -F F -p blastp id="p-223" id="p-223"

[00223] Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10~10, more preferably less than 1 x 10~20, less than 1 x 10"30, less than 1 x 10"40, less than 1 x 10"50, less than 1 x 10"60, less than 1 x 10~70, less than 1 x 10"80, less than 1 x 10~90, less than 1x10" 10°, less than 1 x 10"110, less than 1 x 10~120 or less than 1 x 10"123 when compared with any one of the specifically identified sequences, id="p-224" id="p-224"

[00224] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. id="p-225" id="p-225"

[00225] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306). 561999 41 id="p-226" id="p-226"

[00226] A polypeptide variant of the present invention also encompasses that which is produced from the nucleic acid encoding a polypeptide, but differs from the reference polypeptide in that it is processed differently such that it has an altered amino acid sequence. For example a variant may be produced by an alternative splicing pattern of the primary RNA transcript to that which produces a reference polypeptide. 4 Diagnostic kits id="p-227" id="p-227"

[00227] The invention further provides diagnostic kits useful in determining the bovine SCARBl allelic profile of bovine, for example for use in the methods of the present invention. id="p-228" id="p-228"

[00228] Accordingly, in one embodiment the invention provides a diagnostic kit which can be used to determine the SCARBl genotype of bovine genetic material. One kit includes a set of primers used for amplifying the genetic material. A kit can contain a primer including a nucleotide sequence for amplifying a region of the genetic material containing a non-reference allele at a polymorphism. Such a kit could also include a primer for amplifying the corresponding region of the reference gene, for example one that produces a reference SCARBl, Usually, such a kit would also include another primer upstream or downstream of the region of the gene. These primers are used to amplify the segment containing the polymorphism of interest. The actual genotyping is carried out using primers that target specific alleles such as those described herein, and that could function as allele-specific oligonucleotides in conventional hybridisation, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing. id="p-229" id="p-229"

[00229] One kit of primers can include first, second and third primers, (a), (b) and (c), respectively. Primer (a) is based on a region containing a SCARBl mutation. Primer (b) encodes a region upstream or downstream of the region to be amplified by a primer (a) so that genetic material containing the mutation is amplified, by PCR, for example, in the presence of the two primers. Primer (c) is based on the region corresponding to that on which primer (a) is based, but lacking the mutation. Thus, genetic material containing the non-mutated region will be amplified in the presence of primers (b) and (c). Genetic material homozygous for the wild type gene will thus provide amplified products in the presence of primers (b) and (c). Genetic material homozygous for the mutated gene will thus provide amplified products in the presence of primers (a) and (b). Heterozygous genetic material will provide amplified products in both cases. 561999 42 id="p-230" id="p-230"

[00230] For example, the kit may include a primer comprising a cytosine at the position corresponding to the C-321G promoter polymorphism in the SCARB1 gene or comprising a nucleotide capable of hybridising to a cytosine at the position corresponding to the C-321G promoter polymorphism in the SCARBl gene. Those skilled in the art will recognise that in such a primer, the cytosine, or the nucleotide capable of hybridising to a cytosine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide. id="p-231" id="p-231"

[00231] In another example, the kit may include a primer comprising a guanine at the position corresponding to the C-321G promoter polymorphism in the SCARBl gene, or comprising a nucleotide capable of hybridising to a guanine at the position corresponding to the C-321G promoter polymorphism in the SCARBl gene. Those skilled in the art will recognise that in such a primer, the guanine, or the nucleotide capable of hybridising to a guanine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide. id="p-232" id="p-232"

[00232] In one embodiment, the diagnostic kit is useful in detecting DNA comprising a variant SCARBl gene or DNA or mRNA encoding a variant SCARBl polypeptide at least partially lacking reference activity in a bovine which includes first and second primers for amplifying the DNA or mRNA, the primers being complementary to nucleotide sequences of the DNA or mRNA upstream and downstream, respectively, of a polymorphism in the portion of the DNA encoding SCARBl which results in increased p-carotene levels (particularly increased P-carotene content in milk fat), preferably wherein at least one of the nucleotide sequences is selected to be from a non-coding region of the reference SCARBl gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the reference SCARBl gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention. id="p-233" id="p-233"

[00233] In one embodiment, the diagnostic kit comprises a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in SEQ ID NO:l or SEQ ID NO:3, for example, for hybridisation with mRNA from a sample of cells; means for detecting the nucleotide probe bound to mRNA in the sample with a standard. In a particular aspect, the kit of this aspect of the invention includes a probe having a nucleic acid molecule sufficiently complementary with a sequence presented in SEQ ID NO:lor SEQ ID NO:3 or complements thereof, so as to bind thereto under stringent conditions. "Stringent hybridisation conditions" takes on its common meaning to a person skilled in the art. 561999 43 Appropriate stringency conditions which promote nucleic acid hybridisation, for example, 6x sodium chloride/sodium citrate (SSC) at about 45°C are known to those skilled in the art, including in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989). Appropriate wash stringency depends on degree of homology and length of probe, If homology is 100%, a high temperature (65°C to 75°C) may be used. However, if the probe is very short (<1 OObp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37°C to 40°C), and raises the temperature by 3-5°C intervals until background is low enough to be a major factor in autoradiography. The diagnostic kit can also contain an instruction manual for use of the kit. id="p-234" id="p-234"

[00234] In another embodiment, the diagnostic kit comprises an antibody or an antibody composition useful for detection of the presence or absence of reference SCARBl, or determining the presence or absence of a variant protein at least partially lacking reference activity, or determining the level of expression of the SCARBl protein, together with instructions for use, for example in a method of the invention.

Sample preparation id="p-235" id="p-235"

[00235] As will be apparent to persons skilled in the art, samples suitable for use in the methods of the present invention may be obtained from tissues or fluids as convenient, and so that the sample contains the moiety or moieties to be tested. For example, where nucleic acid is to be analysed, tissues or fluids containing nucleic acid will be used. id="p-236" id="p-236"

[00236] Conveniently, samples may be taken from milk, tissues including blood, serum, and plasma, cerebrospinal fluid, urine, semen or saliva. Tissue samples may be obtained using standard techniques such as cell scrapings or biopsy techniques. For example, the cell or tissue samples may be obtained by using an ear punch to collect ear tissue from bovine. Similarly, blood sampling is routinely performed, for example for pathogen testing, and methods for taking blood samples are well known in the art. Likewise, methods for storing and processing biological samples are well known in the art. For example, tissue samples may be frozen until tested if required. In addition, one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components. 6 Computer-Related Embodiments id="p-237" id="p-237"

[00237] It will also be appreciated that the methods of the invention are amenable to use with and the results analysed by computer systems, software and processes. Computer systems, software and processes to identify and analyse genetic polymorphisms are well 561999 44 known in the art. For example, the results of one or more genetic analyses as described herein may be analysed using a computer system and processed by such a system. id="p-238" id="p-238"

[00238] Both the SNPs and the results of an analysis of the SNPs utilised in the present invention may be "provided" in a variety of mediums to facilitate use thereof. As used in this section, "provided" refers to a manufacture, other than an isolated nucleic acid molecule, that contains SNP information of the present invention. Such a manufacture provides the SNP information in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the SNPs or a subset thereof as they exist in nature or in purified form. The SNP information that may be provided in such a form includes any of the SNP information provided by the present invention such as, for example, polymorphic nucleic acid and/or amino acid sequence information, information about observed SNP alleles, alternative codons, populations, allele frequencies, SNP types, and/or affected proteins, phenotypic effect or association, or any other information provided by the present invention in Tables 1-3 and/or the Sequence ID Listing. id="p-239" id="p-239"

[00239] In one application of this embodiment, the SNPs and the results of an analysis of the SNPs utilised in the present invention can be recorded on a computer readable medium. As used herein, "computer readable medium" refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising computer readable medium having recorded thereon SNP information of the present invention. One such medium is provided with the present application, namely, the present application contains computer readable medium (floppy disc) that has nucleic acid sequences used in analysing the SNPs utilised in the present invention, together with derived amino acid sequence, provided/recorded thereon in ASCII text format in a Sequence ID Listing. id="p-240" id="p-240"

[00240] As used herein, "recorded" refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the SNP information of the present invention. 561999 45 id="p-241" id="p-241"

[00241] A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon SNP information of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the SNP information of the present invention on computer readable medium. For example, sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as OB2, Sybase, Oracle, or the like. A skilled artisan can. readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the SNP information of the present invention. id="p-242" id="p-242"

[00242] By providing the SNPs and/or the results of an analysis of the SNPs utilised in the present invention in computer readable form, a skilled artisan can routinely access the SNP information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. Examples of publicly available computer software include BLAST (Altschul et at, J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) search algorithms. id="p-243" id="p-243"

[00243] The present invention further provides systems, particularly computer-based systems, which contain the SNP information described herein. Such systems may be designed to store and/or analyze information on, for example, a number of SNP positions, or information on SNP genotypes from a number of subjects. The SNP information of the present invention represents a valuable information source. The SNP information of the present invention stored/analyzed in a computer-based system may be used for such applications as identifying or selecting subjects, in addition to computer-intensive applications as determining or analyzing SNP allele frequencies in a population, mapping disease genes, genotype-phenotype association studies, grouping SNPs into haplotypes, correlating SNP haplotypes with response to particular drugs, or for various other bioinformatic, pharmacogenomic, drug development, or selection or identification applications. id="p-244" id="p-244"

[00244] As used herein, "a computer-based system" refers to the hardware, software, and data storage used to analyze the SNP information of the present invention. The minimum hardware of the computer-based systems of the present invention typically comprises a central 561999 46 processing unit (CPU), an input, an output, and data storage. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. Such a system can be changed into a system of the present invention by utilizing the SNP information, such as that provided herewith on the floppy disc, or a subset thereof, without any experimentation. id="p-245" id="p-245"

[00245] As stated above, the computer-based systems of the present invention comprise data storage having stored therein SNP information, such as SNPs and/or the results of an analysis of the SNPs utilised in the present invention, and the necessary hardware and software for supporting and implementing one or more programs or algorithms. As used herein, "data storage" refers to memory which can store SNP information of the present invention, or a memory access facility which can access manufactures having recorded thereon the SNP information of the present invention. id="p-246" id="p-246"

[00246] The one or more programs or algorithms are implemented on the computer-based system to identify or analyze the SNP information stored within the data storage. For example, such programs or algorithms can be used to determine which nucleotide is present at a particular SNP position in a target sequence, or to analyse the results of a genetic analysis of the SNPs described herein. As used herein, a "target sequence" can be any DNA sequence containing the SNP position(s) to be analysed, searched or queried. id="p-247" id="p-247"

[00247] A variety of structural formats for the input and output can be used to input and output the information in the computer-based systems of the present invention. An exemplary format for an output is a display that depicts the SNP information, such as the presence or absence of specified nucleotides (alleles) at particular SNP positions of interest. Such presentation can provide a rapid, binary scoring system for many SNPs or subjects simultaneously. It will be appreciated that such output may be accessed remotely, for example over a LAN or the internet. Typically, given the nature of SNP information, such remote accessing of such output or of the computer system itself is available only to verified users so that the security of the SNP information and/or the computer system is maintained. Methods to control access to computer systems and the data residing thereon are well-known in the art, and are amenable to the embodiments of the present invention. id="p-248" id="p-248"

[00248] One exemplary embodiment of a computer-based system comprising SNP information of the present invention that can be used to implement the present invention includes a processor connected to a bus. Also connected to the bus are a main memory (preferably implemented as random access memory, RAM) and a variety of secondary storage 561999 47 devices, such as a hard drive and a removable medium storage device. The removable medium storage device may represent, for example, a floppy disc drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium (such as a floppy disc, a compact disc, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device. The computer system includes appropriate software for reading the control logic and/or the data from the removable storage medium once inserted in the removable medium storage device. The SNP information of the present invention may be stored in a well-known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium. Software for accessing and processing the SNP information (such as SNP scoring tools, search tools, comparing tools, etc.) preferably resides in main memory during execution, id="p-249" id="p-249"

[00249] Accordingly, the present invention provides a system for performing one or more of the methods of the invention, said system comprising: computer processor means for receiving, processing and communicating data; storage means for storing data including a reference genetic database of the results of genetic analysis of a bovine with respect to one or more milk or tissue colour or p-carotene content traits and optionally a reference milk or tissue colour or [3-carotene content traits database of non-genetic factors for bovine milk or tissue colour or |3-carotene content traits; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic merit of the bovine, said outcome being communicable once known, preferably to a user having input said data. id="p-250" id="p-250"

[00250] Preferably, said system is accessible via the internet or by personal computer. id="p-251" id="p-251"

[00251] Preferably, said reference genetic database comprises or includes the results of one or more analyses of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or p-carotene content traits. id="p-252" id="p-252"

[00252] In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data 561999 48 consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more milk or tissue colour or p-carotene content traits in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with bovine milk or tissue colour or P-carotene content traits. id="p-253" id="p-253"

[00253] Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or P-carotene content traits. id="p-254" id="p-254"

[00254] It will be appreciated that it is not intended to limit the invention to the above example only, many variations, which may readily occur to a person skilled in the art, being possible without departing from the scope thereof as defined in the accompanying claims. id="p-255" id="p-255"

[00255] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth. id="p-256" id="p-256"

[00256] The invention consists in the foregoing and also envisages constructions of which the following gives examples only.

EXAMPLE -Analysis of the genetic basis for milk fat colour id="p-257" id="p-257"

[00257] This example describes the investigation of the genetic basis for observed variations in milk fat colour using the results of a Holstein-Friesian X Jersey cross-bred trial conducted to facilitate the discovery of QTLs, genes and mutations associated with economically important milk traits.

Materials and methods 1. Trial design id="p-258" id="p-258"

[00258] A Holstein-Friesian x Jersey crossbred trial was conducted using an F2 trial design with a half-sibling family structure. Reciprocal crosses of Holstein-Friesian and Jersey animals were carried out to produce six F1 bulls of high genetic merit. 850 F2 female progeny forming the basis of the trial herd were then produced through mating of high genetic merit F1 cows with these F1 bulls. The herd was formed over two seasons; animals in cohort one were born in spring 2000, and entered their first lactation in spring 2002, while animals in cohort two were born in spring 2001 and entered their first lactation in spring 2003. A total of 724 F2 cows entered their second lactations (during which milk fat colour was measured). 561999 49 The animals were farmed under standard New Zealand dairy fanning practices using a pasture based management system. All animal work was conducted in accordance with the Ruakura Animal Ethics committee. 2. Milk fat colour measurement id="p-259" id="p-259"

[00259] All milk measurements were taken during the animals' second lactation. Cows were milked twice daily; milk volume was recorded at each milking. Milk fat colour was measured at three time points during lactation: peak lactation (35 days post-calving), mid lactation (mid November) and late lactation (late February). On each collection day, samples were collected from the a.m. and p.m. milkings and combined to make a single composite sample for each animal. Milk fat colour was measured as previously described (Winkelman et al, 1999). Briefly, nonsaponifiable material (including carotenoids) was extracted from fresh milk samples and the absorbance at 450nm was measured. Fat colour (jig fl-carotene/mg milk fat) was calculated (Winkelman et al, 1999). 3. Genotyping id="p-260" id="p-260"

[00260] Genomic DNA was prepared from whole blood from a total of 1665 animals within the trial pedigree (850 F2 daughters, six F1 sires, 796 F1 dams, and 13 selected F0 sires). An initial whole genome scan was conducted by genotyping each animal for 285 microsatellite markers, obtained primarily from published marker maps. Subsequently, the pedigree was genotyped using the Affymetrix Bovine 10K SNP GeneChip. A total of 6634 informative SNP markers were placed on the map. 4. Candidate gene sequencing id="p-261" id="p-261"

[00261] SCARBl was identified as a candidate gene for the milk fat colour QTL on chromosome 17. Intron/exon boundaries were determined using the UniGene bovine Bt.4520. The promoter sequence was amplified using the primers presented as SEQ ID NOS: 5 and 6. Exons were amplified using the primers presented as SEQ ID NOS: 7-44 and sequenced in both directions. The reference gene sequence for the SCARBl gene is shown in SEQ ID NO. 1. In addition to coding regions, an additional 1.2 kb of 5' untranslated region was also sequenced. The predicted structure of the bovine gene is shown in Figure 3. Primers were designed within introns so that complete sequence was obtained from each exon.

. Statistical analysis id="p-262" id="p-262"

[00262] The dataset consisted of milk fat colour phenotypes collected during the animals' second lactation for two cohorts of F2 animals. Data manipulation was performed using SAS (version 9.1). Phenotype data for milk fat colour was recorded at three time points during 561999 50 lactation (peak, mid and late lactation). These data were matched with the following covariates: cohort (cohort 1 or cohort 2), sire (sires 1 - 6), milk fat%, milk protein%, lactose%, milk solids%, milk yield, condition score, live weight (average taken for ± seven days around each of the milk fat colour time points at peak, mid and late lactation), somatic cell count (threshold of 200,000 cells during ± seven days around sampling times), free fatty acids (as an indicator of milk fat quality, measured in the same sample as milk fat colour at peak, mid and late lactation), calving week, and estrus week. Animals with missing data points for any of the measurements were excluded and the fmal datasets included 597, 648 and 632 observations at peak, mid and late lactation, respectively. id="p-263" id="p-263"

[00263] Analyses were conducted using both raw and log-transformed data. ANOVA was conducted for the milk fat colour phenotype at peak, mid and late lactation. The fmal ANOVA models for each of the lactations were produced using a backward elimination process; all the covariates were included in the model at the first stage of the modeling process and the least significant covariates removed at each subsequent stage until all the remaining covariates were found to be significant (sig. level set at 0.1). Thus, the final models were as follows: peak lactation (sire, cohort, protein%, calving week), mid lactation (sire, cohort) and late lactation (sire, cohort, protein%, milk solid%, and somatic cell count). 6. QTL detection id="p-264" id="p-264"

[00264] The data used for QTL detection were the residuals from each model for both non-transformed and log-transformed data. The raw phenotype data (no covariates or modeling) was also used to detect QTLs. Since the same results were detected with each kind of data, results presented below used non-transformed, modeled data. QTL detection was conducted using a line of descent model (Haley et al. 1994) and a half-sib model (Baret et al. 1998). Subsequently, the SCARBl polymorphism was included as a covariate into the models for peak, mid and late lactation, and ANOVA was performed to test for an association between the SCARBl mutation and P-carotene concentration of milk. 7. Tissue SCARBl mRNAmeasurements id="p-265" id="p-265"

[00265] Subcutaneous adipose tissue biopsies (~500mg tissue) and liver biopsies (-100 mg tissue) were taken from a subset of 40 cows (8 CC animals, 21 CG animals and 11 GG animals). Total RNA was isolated using the Qiagen RNcasy method, according to manufacturer's instructions. RNA yields were quantified by absorbance at 260nm and RNA integrity was determined using the Agilent Bioanalyser. SCARBl mRNA was determined by quantitative real time PCR using the Roche Universal Probe library system. An Intron 561999 51 spanning assay was used to prevent amplification of any contaminating genomic DNA, The assay was designed using SEQ ID NO: 3 as a reference sequence. The details of the primers (SEQ ID NOs: 42 and 43) and amplicon sequence (SEQ ID NO:44) are presented in Table 1 below. Universal probe number 62 was used for the assay (sequence cagcaggt).

Table 1: Details of SCARBl quantitative real time PCR assay Primer Length Tm %GC Sequence Left IS 59 50 tggtgccctcaatcatca [SEQ ID NO:42] Right 22 60 45 catgttgaaagacaggctgttg [SEQ ID NO:43] Amplicon 74 tggtgccctcaatcatcaagcagcaggtcctcaagaatgtgcgcatcgac cccaacagcctgtctttcaacatg [SEQ ID NO:44] id="p-266" id="p-266"

[00266] Results are expressed as fold-change relative to animal single reference sample (this animal was not included in the subsequent analysis). Genotype effects were determined by ANOVA, using genotype as a fixed effect, and the significance level was set at p ~ 0.05. id="p-267" id="p-267"

[00267] p-carotene adipose tissue measurements were determined using HPLC with commercial standards, based on a published method (Hulshof et al., 2006). The inter-assay variation was on average 5%.

Results 1. Detection of a major QTL for milk fat colour on bovine chromosome 17 id="p-268" id="p-268"

[00268] Analysis of the P-carotene data within the line of descent model of QTL analysis showed the presence of a significant QTL on bovine chromosome 17 (Figure 2). The maximum F value for the QTL was 10.6, and the most likely position was estimated at 71 cM. 2. Identification of SCARBl as a candidate gene and detection of a polymorphism id="p-269" id="p-269"

[00269] The SCARBl gene was thought to be approximately located between markers BMS1879 at 50.7 cM and BM1862 at 67.5 cM on bovine chromosome 17. To determine whether the SCARBl gene was involved in the observed variation in milk colour and |3-carotene content, the SCARBl promoter and coding regions were sequenced in the six F1 sires to identify any genetic polymorphisms that could potentially alter the function, activity, or expression of this protein. The predicted structure of the bovine gene is shown in Figure 3, and in SEQ ID NOs: 1 and 3. id="p-270" id="p-270"

[00270] One polymorphism in the bovine SCARBl gene was identified. A substitution of C to G at genomic nucleotide position -321 (see SEQ ID NO:l) in the promoter of the SCARBl gene was heterozygous in five F1 sires. The remaining sire was homozygous for the G allele. To determine whether this polymorphism was associated with the milk colour 561999 52 and p-carotene content phenotypes, the remainder of the FJXB trial pedigree was genotyped. The frequency of each SCARBl genotype in the F2 population is shown in Table 2 below. Table 2: Genotype frequencies of F2 population Genotype Total % CC 194 23.52 CG 403 48.84 GG 228 27.64 Total 825 100.00 id="p-271" id="p-271"

[00271] The SCARBl genotype was subsequently introduced into the statistical model as a fixed effect. As shown in Figure 5, this reduced the QTL significance (F-value), showing a close association of the SCARB1 genotype with the milk fat colour QTL variation. 3. SCARBl polymorphism has a significant effect on P-carotene concentration in milk id="p-272" id="p-272"

[00272] The effect of the SCARBl polymorphism on milk fat colour is shown in Table 3 below and in Figure 4.

Table 3: Genotype effect on milk fat colour Milk fat colour Genotype N Peak Lactation N Mid Lactation N Late Lactation CC 150 11.6 160 9.52 152 7.55 CG 293 .87 320 8.91 298 7.19 GG 178 .22 186 8.81 174 6.73 id="p-273" id="p-273"

[00273] Animals homozygous for the C allele produce milk with greater milk fat colour and greater concentrations of p-carotene than heterozygous (CG) animals. Furthermore, heterozygous animals produce milk with greater milk fat colour and greater concentrations of p-carotene than animals homozygous for the G allele. The genotype effect was similar at each stage of lactation. 4. SCARBl polymorphism has a significant effect on SCARBl mRNA levels in liver tissue id="p-274" id="p-274"

[00274] The effect of the SCARBl polymorphism on SCARBl mRNA levels in liver tissue is shown in Figure 7. SCARBl mRNA levels were measured in liver samples from a subset of 40 F2 cows. The relative expression level of SCARBl mRNA was significantly lower in liver tissue from animals homozygous for the C allele. Heterozygous animals had intermediate expression of SCARBl mRNA, and animals homozygous for the G allele had the highest relative expression of SCARBl mRNA. 561999 53 . SCARBl polymorphism has a significant effect on adipose tissue P-carotene content. id="p-275" id="p-275"

[00275] The effect of the SCARBl polymorphism on adipose tissue p-carotene content is shown in Figure 6. Animals homozygous for the C allele had higher concentrations of b-carotene in adipose tissue than heterozygous animals or animals homozygous for the G allele. Discussion id="p-276" id="p-276"

[00276] The present invention recognises that the SCARBl polymorphism described above is useful as a selection tool to breed animals with higher or lower milk concentrations of p-carotene, with increased or decreased milk fat colour, or with increased or decreased tissue colour or P-carotene content. Such a strategy may allow the production of, for example, milk products more suitable to particular markets, depending on the preference for white or yellow milk and milk products, or the dietary or health requirements prevalent in a market.

Publications Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 411:199-204. Winkelman et al., Estimation of Heritabilities and Correlations Associated with Milk Color Traits, J Dairy Sci. 1999, 82:215-224.

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INDUSTRIAL APPLICATION id="p-277" id="p-277"

[00277] The present invention is directed to methods of genotyping bovine to facilitate the selection of animals with altered milk or tissue colour or p-carotene content traits. In particular, such traits include desired milk fat colour or [3-carotene content, or desired tissue colour or [3-carotene content. It is anticipated that herds of bovine selected for such traits will produce milk fat and tissue of more desirable colour (whether more or less yellow), or more desirable p-carotene content, and therefore be of significant economical benefit to farmers.

SEQUENCE LISTING <110> Vialactia Biosciences (NZ) Limited <120> Marker Assisted Selection of Bovine for Desired Milk or Tissue Content <130> 566948 JBM <160> 44 <170> Patentln version 3.2 <210> 1 <211> 92944 <212> DNA <213> Bos taurus <221> variant <222> (1188)..(1188) <223> -321 C/G polymorphism <221> CDS <222> (1509) .. (1634) <221> misc_feature <222> (27335) .. (27384) 561999 55 <22 3> <221> <2 22> <221> <222> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <221> <222> <223> <221> <222> <221> <222> <221> <222> <221> <222> <223> <221> <222> <221> <222> <221> <222> <223> <221> <222> <4 00> n is a, CDS (49437) CDS (52514) or t . (49594) . (52555) misc_feature (55223)..(55272) n xs a, CDS (57539) CDS {60150) q, or t (57742) ..(60245) misc_feature (60827)..(60876) n is a, c, g, or CDS (62427)..(62542) raisc_f eature (63468)..(63814) n is a, c, g, or CDS (65184)..(65350) CDS (72042) CDS (77621) .(72160) .(77694) misc_feature (79987)..(80036) n is a, CDS (82267) CDS (83183) c, g, or t (82318) (83329) misc_feature (85511)..(85560) n is a, c, g, or t CDS (86998) .. (87123) 1 gttgaagaag aatttcattc tgagccctca gggactgggc tgccttaccc aggtgttggg 60 gatccccgcg tgaccctcaa ctagacaatg aggggactga cctacatcag ggttccagga 120 tcagtgcagg gtgggggtgc tgcgggcact ctgagcaggt tggcgggggg gcctcatgga 180 ggactgcagg ggagcacagg cccatgtttt actattttgg agggggtgtc agtaatctat 240 ccttcatttc ctcacttgcc catcctctgt cttcctgttc attagaaagc agacacttgg 300 atttgttgac ctaacctgct gtgcctgttt gcttatgtgc aaaatggagt cggtgtggga 360 aaggctttta gcacagaacc tggctcccag aaggctcctc accgcttcat tctaaactgt 420 tccttgtgag cagacattcc tagccaggca gccgtcaagc tactgagagg ggagggtggg 480 ggcagagagc tgccccgtag cattttaggg cctggagatg gtcacaatgc aagaacaaat 540 gaatgtggca catgcctggt ggcgcccctt gataagggtg aggggagagc tgacgctatt 600 ctatttcaca cagggtggct gggaaggcct ttccgataag atggccccag cagtcgtgaa 660 ggaagaagcg gggcagatat ctgggtgcgg gttgggggcg gacagaattc caagttgtgg 720 ggagtatgag gtcctgaggc aggagtctgc taggcttgtt ccagggagaa agaggctgga 780 atagaatgtg tgtgcacgtg tgtgtgtgtg gggggggggg ggctgggatg gggtggggcc 840 agctgaggac gcagaggtgg gtgcggggtt ggggggggga agctctggag ttcctgcaag 900 agttgggaaa tttctactaa ataccaaaaa tgatgacggt caggacagag gaagtgggag 960 gggaggaggg gcgaaatgag aggaggagga ggagggagac agcgggagga gggggagaga 1020 gatgcggggg agggagaaga tgggggtgct catggaaagg gataagcagg gagcaggtgg 1080 gagtgaggaa gcggggaagg gttaggaggt ggaggggagc caggtccaac tttgcacgcg 1140 ggccacgagg gccccagggc tccctcagcc agaaacggaa gctcgggcgc cggattcccg 1200 gcggtggccc cgccccagtc ggaccgtctg atccgcgccc cgcctcgggc acgccccttg 1260 561999 56 ctaacttagg ccccgcccct tcgcccatgc tcgcccgcgc cggcccgaat cccggtgccc 1320 agggegtccc ggggggcggt ccgccccggt gatggcgcat aaaaccgctg gccacctgcc 1380 gggccgctcc tccgtgctct gcgggcctcg tcctcggagt cgctgcctct atcacctccg 1440 tccccgtctc ggtccctcca caggcgcgga accccgcgag caccgcgcgg gcgccggggc 1500 gcgcagac atg ggc aac ctc tct aga gcg cgc egg gtg acc gcg gcg ctg 1550 Met Gly Asn Leu Ser Arg Ala Arg Arg Val Thr Ala Ala Leu 15 10 ggt ttc ate ggg ctc ctc ttc gcc gtg ctg ggc att ate atg ate gtg 1598 Gly Phe lie Gly Leu Leu Phe Ala Val Leu Gly lie lie Met lie Val 15 20 25 30 atg gtg ccc tea ate ate aag cag cag gtc ctc aag gtgggtgagg 1644 Met Val Pro Ser lie lie Lys Gin Gin Val Leu Lys 35 40 ggcttggcct gggacccatg cgcgggatac cgtccgaggc tggagggcgt tgggcgcccg 1704 ggaaggccag gcgcgcagcc ggcgggctgg cgacccggtg ccgggatccg cgtgccctgg 1764 gcccgggccc ctgagcaccg gagagaccag agggcatgag gcaagccagt tggccatctt 1824 tcccaggaac ctccccgact caggacgcag gctgagtttg ggggccaagg aggcccccca 1884 gggccttctg ctggaggcac tggactgggg ttgggaaagc ctgaaggaga gaccacctgg 194 4 gagcctgacc tcacctgaag ggttcggggt ccaaggcggg cgtggagtgt ggccgtgttt 2004 ctgatgccgc caggagaagc cctggggcag actccatgct tcttgcctac tgtacagcag 2064 cctctgacgg tgtctggtag aactggcact geaetttget tgcttcctgt gagcgcaggc 2124 ggacaggaac cctccaagca tgtgcatgtg aggctcacat gggtgcccag gccgccactt 2184 gtccctgctt ccaaaacctc cccttgcaga cctcttcttt aaaggcagaa tgggttgccc 2244 tctcctcccc tgccgctgcc ttcctgatgc agattggcag tgataagctg ctttactccc 2304 cagatccacg catgggcagg aataagccca aggctgcata aacagggtct ttcaggagcc 2364 cacagcctga gggttttcct ctgatcctaa ccttatacac cactgccctt tcaaccaggg 2424 agacaaatta aggctgtcgg ctggccttca gaccggcaga ctctgccctg ggtgtctggg 2484 cagcctgttt gaatgagcct ggagttactg aaggacaggc agattccaga gtatcacatt 2544 caagetaaaa ccattcaggg etttgaggee aaggccacag cacctgcagg gctcagaggg 2604 gaaactgagg ctcaccttgg cgcctcccag atacagccaa gatgeagage tgggataaag 2664 atgtggctct tgtcacttgc taattgattg ctctttcccc ctgctttagg cgagtttctt 2724 gagaatgagg actgggtcac atttcatgtc catgccccat gcccaatgca agatgettga 2784 gagatgaatg aatgatgaat gaatgaatga aggctgtatc tgcttagacc eggttgccat 2844 ttttttggct ttaaagatgg agcaacaccc ttctggtttt tctcgaggtc tctgggccag 2904 tggttgtctg ggtcatattg gccacagcat gtcctttgtt ttgttctcaa atcttgtccc 2964 attaagtcct gtaatctgat tgcagggagg tcctggccaa tggtgacatt gagttctgag 3024 ttcctcttag tggtttttta attttgcctg tgaagtggcg ggaaaaagtt ttgtggggag 3084 acctcagagc aacagaactg cttcttttta tcccatggcg gccaatgggt gtttttgett 3144 tgcttttcaa agtagctgta agaaaaaaaa aaaaaaaaaa gtagctgtga gagctttatc 3204 tcagtgaaag ccggctgtaa acgcctgcca attttgtagt ttctgtgctg tgtgtaccct 3264 ggagggatgg tcactcgtgt ctgtatgcat ggtgaaccct gtcgtgatcc tgtgcgagag 3324 cctcttactt tgggggtggg gggatgggga atgagtggaa tttatagttg atgaagaatc 3384 ttgtcaaccc agcccttctt ggacttaatg caggtgtttt ctttttatgg cagtcagaag 3444 gatgtgtatg tgtttgtgtg aatgtacatg cgcacatgca tgccctacct aatttctcat 3504 tcctcctctg ggcttccaga gaagaaagca ccactgtcct cagttgtccc ggagctaaga 3564 tttccaggct taaacttgag ggagagtcac ccagcatcta ccataaatat cccaaaggtg 3624 attggtctgt ggctgagtgg tgtgaagaca tggaggegag cccagcccgt gccagcttct 368 4 tggacagctc aggegaaget ccggccaaga gccttccgtc agaagaagag tgtgtgttca 3744 ggagactgag tgaatagtac attaggagag gaagggatgg cctgttggtg tttgctttgg 3804 ctgcacgtgt ccatgtacaa cttgaatggg aggacttgtg ctttgatttc tactgtttta 3864 tgctttgggg ttaatgaaag aagtccatgt atataaaaga catgeattct tttttaaccg 3924 agaaactatt tctcttcttt tttaaaatat aattttattt atttgttttg tggctgtgct 3984 ggctcttcac tgctgtgagg gcttctttct agttgcggcg ageaggggee cctcttttcg 4044 gtgeaeggge ttctcattgt ggtgacttct cttgttgtgg agcaggggct ccagggcact 4104 cgggcttcag tagttgtggc atgtgggctc actagttgcg attcctggac cetagagcae 4164 aggctgagta gttgtggtgc acgggcttag tggctctgca gcatgtggga tcttcctgga 4224 acaggggtcg aaactgtgtg tctctgccat tgacaggtgg attcttcatc actgagccac 4284 561999 57 cagggaagcc ccaggacatg cattcttgtt tgtaaaaaaa attcaagttg agggaaaaca 4344 gggtctctcc cctcgctcat tttattcccc ttctcgatag taccactatc atcagattat 4404 ctttgtaaga agacttttat agagatacac atcatggatg tgtatttaga catagtaata 4464 tatacttaca cgtattgaac tatactatgt ctaaacagcc tgcagtttgc ttttcaattc 4524 acagatacca tttgaaaatg tttccatatc tgtcacattt tcactgctgc atagtatttc 4584 tttgaatact gtacacttac ctgtttctcc actgactgat aattttctca tacatacctt 4644 tctgtgtatg tggatcacca cttccactgg gtgcatttct agaagagaaa cctctagacc 4704 agaaggtctg tccgttttga atttggtcac agcctaatga cctttctgaa gatcaagagc 4 7 64 atttataatt ccatccagag tatgaaacta ctcactcccc cacatttctg ccattattgg 4 824 tgatgaccag tgtattagca tcttgaataa ttttggcggg ttaatctgat gcgtgtaatt 4884 tacatttctc aggttgctga tggggttgca cccgttttga aactggtgtc agtttccaga 4 944 gctcagggtt tgcagacctg ggcgtgccct ttgcttgctg gggacctcag gccaggtact 5004 tcacctctga gccaggggtt gtgtcttcag ctctgaggca ggaatcagat ctagtttgcc 5064 tcctgtcaag atgatgaagt gcaaaggcga tgacatgtgt gtaaccctgg atgtacccgt 5124 tttcccattt gaaggttatc acactcatgt aagctgttgt ggtgtttttc cttcctacag 5184 ataaggaaac tgaggcatac aaagcccaga gacatgtcag ggttcacaca gctcatggca 5244 gttatggtca ggaatcctga ccacaaatct gggtctacac ttcaataggg gaataggaag 5304 gaatgcagaa gtcttttatc cagtggattt ctgggcagag attatagaga aagatcttta 5364 tatatttggg cacagaaaag caaagcggac gaacatgtga ctaattgaaa aaagccataa 5424 ggttctttgt gttaaaaaaa aaaacactca atgaaattct gtatttttac tagatttcta 5484 tcagtgcttg taaaagcacc gaaaaaggtg tggaagggtg caccagactg acaaaagtgg 554 4 ttactcaggg aactgggggt gtggtggggg gaaacatgct ttatctgtga agttcatact 5604 ttttttttta ataattaatt aattattaat ttgtctgctc caggtcttaa ttttggccca 5664 tgggatcttt ttttttttca gttgcagcat gcagagcttt tttagtttcg gcatgcaaac 5724 tcttagttgt ggcttgtggg atctagttct ctgaccaggg attgaatctg ggttttctgc 57 84 actgggagtg tggagtctta gccacaaacc cccagacttt ttctttttaa acaaggagaa 5844 tatattcatg tattatgtgt ttagttaaag gttagttatg aatccatagc tttcccccaa 5904 agaatggcca gatgagttca atacagagtg tgggaggctc acagctgagg ggacctgtgg 5964 atggagctcc ctgggagagg ccttgaggga gaagaagact catttcttct cctttctccc 6024 cgccagtggg gagggttgga tgagaggtat tcagactgaa cacatttagg agtgtaatca 6084 aggatgacta cgtgccaggc atcttggtaa atgtcaatca tctcaattag tcctgggaga 6144 attctgggac acacctctca ttgtcatcgt ctacagttag ggaaacaaaa gcttaagaga 6204 gtaatttgct ggggtgtgtg tgtgtgtgtg tgtgtaaata gtctgaggca aatcccagga 6264 ggacaggcat gcttgtatcc atagcctcga atggagcctg gcatgcgtgg ccctctctgt 6324 atggaccagg aaccctgggc taggtctctg ggcatgtatt cccaaattac tggctgtctc 6384 tggtccacca ggggaggtcc tgctgggcca aggggaaggg gcttatctgc tgggccactg 64 4 4 cgtgtagtga tggacctggt agccttctcc acggccgggg gtgggggaag tgggaaaccg 6504 aatctctctt ttgtaaaaaa agattttatt tgttttggct gtgccgggtc ttagttgaag 6564 catgtgaaat ctttagttgt ggcatgtggg atctagttct ctgaccaggg atcgaacctg 6624 ggccccctgc attgggaact cagactgtaa gccactggac taccagggaa gctccttctc 6684 tgccatcttt aaagttttat agttggaatc agccttgggt gctttatcca ctcatttttc 6744 cagctgccaa attcttccct cacctgtgtc cccagctcaa taaatgacac ctccatttgt 6804 tccctcaagc acaaaccttg ggttatcctg gatgcctttt gtggtatcct tgtatctgct 6864 ccatcagcaa aatctattag gattaccttc aaaacatatt tgcattctga caccttgtca 6924 atatctttgc cactctggcc ttctccattc accacttgga ctgatgctgt agtctcatct 6984 ctggtctcca tagacaacct gatcacgtgt cctctctgct cacagccctt taaggtatcc 7044 ccatttcact ctgagtgaat accaaggtca gtgtggtgac ctccctgcca tgatccagct 7104 cctgttgcct ctttcactgc accccctgcc ttggcccctc tgctcacctg ccacagtgtc 7164 cagtcttcag tgtccttgag cccactgagc ctggttccac caaactctct ggcttccttt 7224 ccccaccgct ctgaaggctc gccccccaga tatctgcata gctcacctct cacatcccta 7284 aaagccaccc cccacttatg ttacatgttg atttgtgcat tttctttctc cctcgtgggc 7344 aggagtgggg gctctcctgt cactctctct gtagtgaccc caggaccaga tcagcgcttg 7404 ggttcacaat gggatggtgg gatgaacaga ccctgagcgc tttgaggttg ggctcttcct 7464 tggccttttc ccacccccac tctcagcctc cctattttta ccccctctcc ctctgcaagg 7524 gccacccaag gaacctttag gacaccatgc tgtgtcccag ttgcttctca ccaaggcgta 7584 agtgctcagt cgtgtgtgac tcttggtggc cccatggact gtagcccacc aggctcctct 7644 gtccatagga attttccagc aagaatgctg gagccagttg ctgtttcgtc cttcagggga 7704 tcttcttgac ccagggatta aacccacatc tcctgtgtca cctgcactga caggtgattt 77 64 ctttaccacg gaatcacctg gaagccatca gttgcttttc gccctctggc agatttcttt 7824 cttgtgtcag ttcagaggtg ggggttgggg agagataacg atcatagaca ctgattaagc 7884 561999 58 accagatacc cttctaggtg tttaacttgc agtcattcgc ttcatttagt atctaaatag 7 944 cttcctcatt ttttattgag ggcaatggaa gcacagagcc gaaaagacac ttgccgcagg 8004 ctgcaccgca gcagcggcag ccccagattc tagctgggtg ggctgctgcc tgtggcccct 8064 cttccccact ctccgtatcc ctcagagctc aacttggtgt ttggaggcag ggcaaaccct 8124 tcccctgctc tgcttgggtc tctgcttggg tccaccgtcc cttgtccttt tcctgcacag 8184 tggggcggtg gggtccttcc caggcttttt tcctgatcat cacctccctc aaactgtgag 8244 tgcagggtgc ctcccgacgc cttccctcca tggctgtccg ataggcctgg gtgggctcta 8304 gaatcaccaa tgccaaggga agctgcctgt ggtcccagcg attctcaggg tggccctgct 8364 gtcctgcagg gtctgcttgg ccagctctgt tccctgtccc ctgtctgggg ctccagaaag 8424 ccccaggggt cgtgtctcag agggtgtcaa agtgttgagg gctgactgca tctggctgcc 8484 tctggccatg gaggataagg ctgtagactt gacctgtggc cgcagatcgc aggggatgca 854 4 ctcccttctc agggggcagt ggggtcgcag gtgtgttcca attttgttgt gtttttcttc 8604 ctctttcctg cctggggctt cctacccctc tggtgtgtga gagggcggct tccactactc 8664 cagagccagg gctgaaaaac atgggctatt tttagtttcc ctgagtgtca gttatttttg 8724 tttccctctt tgcttctcat ggatttgctt caccagtgtg tgtgtttgag agtgtgctgc 8784 atatacatgt tgatgtatgt atacgcgtgt tggtagtaca cgtatgacac atgaacatgt 8844 gttggcacgc acccacgcac acttgtgtgt gaccatatgc tgaagcatgc acacattcat 8 904 gttgtgtgtt ggcatggggg cacagtgtct ttgtgatagc atatgtatgt atgctcatga 8964 acacatgtgt acacacacat tgacatgtgc ccacacttgg ttcttggcac acgtgggttc 9024 acacgtatta ttgttatcca tgtgtatatg cgtgggttca catgtgtgtc ggcacatgct 9084 gggcgtatag ttacacatgt ggacacctat ggtaacacgt gttccaagcg tgttggggtg 9144 tgtatacaca cacgtggcca tgtgtcgtat tgcagtctgg ctttctggag gcctggcccg 9204 gggctgtgtt cagtgcacct aaagaaagaa agaaagaaag aaaaagtgaa gtcgcttagt 9264 cgtgactgac tcttgtgacc ccgtggactg tagcctgcca ggctcctcgt ctttggaatt 9324 ttccagggaa gggtactgga gtggattgcc atttccttct ccaggggatc tcaggagggc 9384 ttccctggag gctcagatgg taaagagtct gcctgcagtg tgggagacct gggttcagtg 9444 tgcctggctt ttcccatttc atgacatgtt ccccaagcct tcctaggatg actcatactt 9504 gccaggcctt ctctctgtga cccatgtggt tagctagaat ttagagaagc ttggctatct 9564 ggaatcatcc tgttatgact tcaacatgta gcaaacggga tggaagtgcg tggatgtcaa 9624 ggacttcctt gttgaagatt ttcattttcc cttgggcagt tttattctgc tggaacctgg 9684 gcccaggcac gtctgctcca tcagtgactg tggatcttgg tgcctggaag catgatccta 9744 tgtgcttggt cagcagcaga gaggcccctg ggcagagagg cccccccaag gcaggtttca 9804 gccacaaggc tgacttctct ggctcatcat cttagaatta gagaatttgg ttacgagagc 9864 atctactccc cttcccccca cccctcttct gtgccataga gatccagcag ctttgtcccc 9924 ataaacacat ttaaagtgca aagaattcaa ctctaggtag gactcagcca aaaaaaaaaa 9984 aaaggagaag ttcttttatg tacctcatct cctacccgct ggttcagatg tgaccttggc 10044 ctccactggc ccttggaggg gccatgggag ctgaaatgca aagagaaaca aaaaccaaca 10104 ccaaaaagct cattcttggc tttggggctc tctggggccc tggagctggc gaattacagc 10164 ctgtccaggg gtgcctggga cccacatttg aagctcagag tcattctgct cagaacctcc 10224 ctgaatccag aggtccgggc ccctggtgac cctgggcaca gctggcttca ttgtctggct 10284 tccttccccc acctccaccc catcgtttcc tcctgtgccc cgatttattg cttcgtgctg 10344 ggtaggagga aggaatagga gtggcccaac ctgtgcttgc ttcctggcat ttggggtgaa 10404 gttccttcca ccttttctgg aagctgctag aaggactctc cagacgcttt gatgtgtggt 10464 ctcttgggct gctcttgaac tcagatgatg gtgtctcctt atctccctga ccgctggcca 10524 ttggcacgtc cctgcactaa agcctttcct ggctgaggtc acacgatctc acacgtctaa 10584 tgccatcttt gtctgggccc ctcccaaatt tatatcctag gcccaggcct ttccccttga 10644 aotccagcct tgagcatcca gtggccagct ctgtatctca gcccgggtgt tcaaaggcaa 10704 gtcatgctta ctctagccag tgcttgactt cgcatcactg cccctgcccc agaacttcat 107 64 tcctcccacc acgttcccat ctcaggccat cgcttctcca tctgccagct cccccggccg 10824 aaagccttgg agtcagcctt gattctctca cttcccacgt gggacctttt gtttttggcc 10884 tcatcttcaa aatatatgca gagagacctc cctggtggtc tagtggctaa gactccacgc 10944 tgtcaatgca gggggcccgg gtttgatccc tggtcagaga gctagatccc acagggcgca 11004 acttttaaag ttcacattcc aggacaaaag atccctcatg ctgcaattaa gagatttcgc 11064 atgctacaac ttagaaaaaa aaaaggtccc tcatgcccca atgaagatca aagatcccct 11124 gtgccctagt taagatgtgg aacagctaaa taaataaaca gataaacgta agatactaaa 11184 taacccgaaa aacacaaaag taaaaatgta cccagaatcc agcttctctc cacacccagg 1124 4 gccaccaccc tggtccaagg caccatcaag tttcacctgg attcctgcac atctccgtgt 11304 ccacctttgc tccctggcta ggtttttcag cacagcagcc aaagggatcc catgaaatct 11364 gcaatcagag catcagcctc ttctgcttaa ataaagcccg gcagtgggcc cacaccatcc 11424 cctccgagtc aaggtgaggc cttatgttgg cccttgaggc ccgtaccatc cgaacctcac 11484 561999 59 ccctttctgt ctaactggca gacgtgtgcg atgaatgtct cccctaccag gccaccaggg tacgaacttt cagaattcac aggcccatga gatgcctagc gagtggtttc gggggctccc ctacaggagc ctgccacagt ctttctttct acactcctgg cacttatgta tgtctctagt tctcattgag agttgtggcc gaacccacga ccctatgctc tgggtgtggt tccctgcact ggttacgcca aggcagtgaa ttcttttcct cttctcccca tcaaatggta cctatctgtg atactgttca gtatcccatg ccgagccagt ctttggcttc cctgggagcc tgcttgtatt gttggtgtct agttgtcact gggcccaggg agggatcctc tatttccctc cttgtcctcg ttgctggtga aatcatttac gcagagaggc aagggatcca gatggccacc caaagggaac taaagttgta ccctgagcca gatgcccgtc ggtggggtgg agggtgacag catctcattg ctaccttgga aatgtttcat gaaacaaaaa gcactgtcag gggagcaggg caggtagaaa cctccccttc catcagcctt tgattctctc ttcttttttt ggatcgaacc aagtccttct ccttaccctg ctttgtatcc gggcagggac acatagtggt acagggaggt ctccccctgt tccctctccg agaatgttct ttggctgtac aagtggaagc tgtacgtact tgcagggagc gtggcttctc cacaggctta tccctgcatt ttaaaaataa atgaaaccca tctgtctgtt cgcagcttgc agcatggagt cccatgcaca gaagcctttt ctcttgatga ttataagcat cttggttgag gttgggaacc gagaggagtc aatgetggct tggtctttgt ccagagatat tctgattgat ggggccatct aggtgtggag ctgctggcat ccactgcatt tgggcagaaa tatgtacttc ctgcactcaa ggtggggagg gcctgaatgc tgcgccacat tttatggagg gtatgaaggc atcactgtag cttggagttg gtggtgccct gcagcagaaa cctcccactg ctgcttgaga aaactcagat cccagaggga ggaagactgt cttgggagaa gttcccaggg agcttgacct attgagttct tcctgcctcc ttttttttgg tgtgcccctt ctttttgaat ctgtttttct tgtttattgc cttgggtttt aggttctcga acgtgaccag gtggctccac ggccgtctgc ttattgtttg ctcacagcct atgttgtctt ttatctgttt gggggctgct ttgttgtggg gttgccatgt ggcaggtgga tgtaaattgc tttgctcctc gtagcctcac aggatcttag cctaaccact gatgttcttt tcctcctgac ttacccctct tccatgttat gttccccggg ctggtctagg caatcgggat cttcccctcc ggtgtgttca ctctctgact ccctcctctg gggggtggta gctggcctgg cttggcttca gaggccctgg tggcccacgt ctctgtacat taaaccacaa gagcttcctg aatccaagag gggggagaaa gaccttcaga tcatgggctc cttgagaaat gagaaggagg aaaggaaaag tggttgatgg cctctgccta gggtggtaac tgttagatct gctaggattt tgtcaagtgg gcccagtggt tcctggtctg tgcacccggc tccctttgcc tttaggcctt tctcactgtg gtagtgcaag attgcaagtt tctgttgccc ttattgtctg gttcattgct atagttactg tggcaagttg tccacatgaa tcctactcac tcttcttctt gcggaacttc aaccactaga ctggctgtac ctctagctgc gcttgggctc ggcatgtgga ttcttattca atcatttcac atggcctgtg agacttttct ttccccgacc ggacaaccaa cctgcctcag ttttatgctg cctcccccac tatcttctaa acaggggcct gcagagacct cttcaaattc ttcccgtcct tggattcagg ccagtgactg ccccaattcg ggttaaaaaa agccacctca gctgggtggc gtcatcatta gaggctggag ataggatctc gtcaactggg tggccctgtt ttaaacattc ctggttgctt ggtaattaga tgattggcag ggactgctct cctttttccc ggtgggaagg atgcttttct ttgtagtgtt taactgatca ggcactcagt aagtcaggcc ggagggaaag tgtgaaacca tatgcctgcc cctgctggct cacagctctt tgttcggatc cagcatgcgg tgtggattct ttcttccctt catcatccgc tttctctcca gtgtcccaag aatggatgtt tcaccctcta gctgtcctat gccactcttg tgggattatt cttgactagg ccaccaggga tgggtcctca ggtgttgcag tagggctcat atcttcccag ttggaccacc aatactgatt agcccctttc tcttgttgtt agggatcgaa agaattcgcc agcctttgca gattcttttc tccccaccct cctgtcacca cgtctgtctt ggctgggaag atggatttgg atctcccgca acctgaatct ggtgtttttc aagggacatc cagcacctgt ctctgcctcc ttggattggg aagtggaatt ttgggagacc atgtcgtctc tcagtattgg gtttctcctg atttttaaac ttaatttccc gaattttccc ggcttgtctc gattggtcaa ccctaaagca actgttagga ccagaatagc agagattatt gctaattgtt tccatctttt agagagcaaa tgggtgatca agtcatgagc cacccacctc gttcctcaga tccctgatgt tcactttctc gaccttactt taaccatggg tccccggtgc tgagatactg tgagagtgtc cacttggaac tagaaaacga tggtgacgtg ctctcaccta tccattttcc tttgatattt gattgaacct agcccagctg ttgcgagggt cgtaccagct gggcttcagt accagggatt agggaagtcc aaaaccttct ctgcatccac ttttgttttt cctgggtcct tcatgagctt cagactgttc atttttaagc atgaacctac ttctggaatg gttcatgact ggaccttagg aaggcagact ccagagagga caacaagtcc ctggctgagc ggtttatgaa cttggcccag cagagggaga ggaggggtgg gtttatgttt caggtccagt aaccatgcaa cctggagggt tgggtcctgt ctagggctca catgagtctc agtggccggg atatgcttat ccttggatta gggagactga gaaggaggaa tttctcactt ttctttgtct tctgtcaggg attctttgag ttaggagtga gatgggctgg taatgttttt ctggaagcag 11544 11604 11664 11724 11784 11844 11904 11964 12024 12084 12144 12204 12264 12324 12384 12444 12504 12564 12624 12684 12744 12804 12864 12924 12984 13044 13104 13164 13224 13284 13344 13404 13464 13524 13584 13644 13704 13764 13824 13884 13944 14004 14064 14124 14184 14244 14304 14364 14424 14484 14544 14604 14664 14724 14784 14844 14904 14964 15024 15084 561999 60 gattgggagg tgcaggcttt ggaagagaga ggagttgacc tcaacagtcc catacccttc ccagggtctc ccttccacct tgacatgtca ctggccagag ggcagcctgg acttaacctc tatacagggc cagccttttt taagggggat ttttcttaca ctgacttaga accttataca cactatgccc caggcacctg gccattccca gttttgggaa ctgatgctga gttgcttgtg agaaatattt tgcttctttg ttacaacaat tttctcctcc aacactagtg ttgtctcttt tgtttgagtc cctctctcac gtgagtttga ctcttgcctg gaagagtcag ggaaatggca tgggctgctg gcagttggac catctgcttc cttccagccg catggaaaca ttgcaactct ttgtcttccc cccatctggc actgccgctg actgcctgtt actttgaatt gctcagtagt gacctgggat cagggaagtc tataatatca ggggctggtg tcagaatggg tacaatattg attatatact attagtgtga cagaagcttg gctttatctg tattctctca aggcctttgg aggtgtttgt gtctttcccg taagcgaagt gggaatcatt cccggatgaa ttcacctagg tgggagcatc gaatgagaag tcatcaaggt ttttggcatc gttcaaatcc tctgtgcctt tcttatgaag ggcaccaggg agttttgaga tcagctccac ataacagctt cacatccatt actttccagc ggtagctctc tgagcagtag agttgtgaac caatttgtag aacgtgatga ttttttgtaa atctcatgct tttttgtctt tctaatccag tttataaagg ctgcatgaac agatgggagt ccccagcttg atcgccccac gaaaatccca acacgactga acccaatcca tctatggggt tggaagaagc ctaatttcct ccctcagcca tgatgccttg ggggtgccag agcctgtccc ctcttggctg gaatcccttg cccagggtcc ttgttttttc tgcagtgcat cgaacccgag ccctttcttt tttatgaaac cactgggacg gaatacatgt taaagttaaa tgaaagtaag tcatctccag cttggcttct attgtgaact aaattcatac gaggtgatta tgtaaactgg ggtgggagcc ttctcacctt gtagctagca ctcctgacaa acagtgagga cccttgcaca catgggacag caggcatggc caggagctag tggttctgcc atttcctcct atggggtgtg actggttttg tgattcaatg ctcagatcat ggcaagtgtc gtaggattag atettgcttt agtgggtatc tgttcttttc acagtgcagt catttgcact catctcactc ccgccagtca cagatgacat tattgtttga actagtcctc gataggccag gcacgttttc agagccctcc gcagccagcc ggactggaag tggacggagg gcgacttcac gtgttcttgc cgcacagagt ccctgattcc tagctctgat gcctgcccgc tgggctgggt agcctgcctg tctgagcctt gcagatgcag gcagctttga gcggattcca tgcgtttctc gggcttagtt tcccctgcat ttattgtgaa gagtcgccag acccaaaggg atacctgtgg ttaaataaaa tgtctaagac ataaaaaagc tatcatgtta ttaaatgagt tttgaaatcc gcttatgagg gagaagtgtc aaccttggtg catcagaact ggtatattct ccttgttgat ggtcggtgtg cagagagtgc actggaacag ttctcaaacc gtggacccaa ctttcttagc ccgtaaaatg ggcacgtgtg tggaagacat catcacgttt ctggcattag acctgagttt ttagttggtc tctgactcca tagatgactg ccaccctgat gaatttctgt ctctgtctct tggagcactg ccgtcacact tcagagtctc atcatgtcct cagccctgtt tgttttggca ttctgacaga tggctgagtt ccctcctgca cggagaaggc agcctggtgg tttcactttt ctggagaatc cggacacgac atctggcctt tccttgccct tcccctccag tgccgcctca gtggagatgg acagaagttc gtcactctgg aagaccaact tctgctgcag ttgttgtggc gcccgaaagc tggaaggcgg atattactgg tccaggttcg atggtatggg cagattcatt ttaaaaataa acacatgtta agaacctcat tgatgtctac ggaatcttac taatgtccaa gtggaaccct agggctgggg tgcactggag tgcccaggta gtgaaccagc ccttgtccca ggagtgaccc cctgtgtgcc ggggagctgg cccaggattc gagcgtagac tgtatgtctt gaattaatga acgtgcttag tttttccagg attgtgcact atcttgaagg tttctcttgt actttaccga tctcttctct aacatcccac gtttcagttt aagccttctc cgtggccaaa cagcgtgcgt caggaaagct ttactgtgcc gtgtctggtt ctttttactg ggatgccctt ttcctacaca gggatggata gccagcctgg aatggcaccc gccacagtcc cactttcatg ccagggacgg tgaagcgact ggctctttca cacgcccagg ccctcatcgt tcctctggcc gatggcaggt ttgtgccagg atttactcgg aggcctcggg tctcctgctt acacagactc gtgtgggatc attcttaacc gagaatggca atgcacgata gagggaggag ttgatatatg ggctgataaa aataaatatc ctgccctcca atccctaaac tgctatgatc agtgatgaag catgaatagg tgtgccaaag ggggtggcgt cagatgcaga acacctgtca caccctagga acctggacag cgagtgagaa caggcagtgt tctctagggt ttcggagcca tggacaagtg aagtatctcc aacgtgggcc gacccaggtg ttatttctgt ttggggaccc catcatatta aatgggactg gaattttccc ggttgtacca gaaaactttt tcgagctcca ccatctggga gtcttaggac tgtgttggtt aagagcacta gccttgtttc aacccttcac caatttggat aaagtctcct tcgggacttt gcccccccgg ctctccagca atggggtcgc cactggagaa gggagcctca taacggctta gctgttcctc tgtgtggatc ctcccaggac tgggaaggca gagggccttc tccccctgac ctgagagccc acttccttcg tggattttca tagagcacag ttagttcccc actgggccac ttgaaatatg ctggatgctt ggaggagggt gcaaaaccaa aaaaagaaat taagataagc gaagcccctg actgggatta tgacttcctg ttaggaggtg attagtgccc 15144 15204 15264 15324 15384 15444 15504 15564 15624 15684 15744 15804 15864 15924 15984 16044 16104 16164 16224 16284 16344 16404 16464 16524 16584 16644 16704 16764 16824 16884 16944 17004 17064 17124 17184 17244 17304 17364 17424 17484 17544 17604 17664 17724 17784 17844 17904 17964 18024 18084 18144 18204 18264 18324 18384 18444 18504 18564 18624 18684 18744 18804 18864 18924 18984 19044 19104 19164 19224 19284 19344 19404 19464 19524 19584 19644 19704 19764 19824 19884 19944 20004 20064 20124 20184 20244 20304 20364 20424 20484 20544 20604 20664 20724 20784 20844 20904 20964 21024 21084 21144 21204 21264 21324 21384 21444 21504 21564 21624 21684 21744 21804 21864 21924 21984 22044 22104 22164 22224 22284 561999 tttagagaac cttccataat aagatctcag gaaccaccca ttaaaaacgc gtgcttactg ttccctggtg ccctggttgg aatatatatt agttgttgag gtgaggccac ccatgtttgt tggtagaatt gctctggaat actcaaaggg aacaaaaacc gacaatttga agccagattg atttttcagg ggaaggacca gcccacgttt caaacccagg ggctaccccc catcagccca tttgggggtc gggagtgcct tttcacaggt tttttttatt gaatcagcta cactcatttt cttgtgagag tggccccccc aggcttttgg gccctcataa gagaagtctg cttccagcct ttgttacagc tttgtcttgt cacgccccat cgagcgtctc ctgatttcct ttccgaagtg gaggtgaaat gcacattcac atcctttctt atggatgtat tgccaggctt agcgctttac tgtttatcca tggtgctgct tttaaaaaaa tctttagtgc ctcgggcttc ataggcttag cctgcattgc tatatttctt ccctgggggg ggtctgcttg cctggctctg gttagactcc ccccctgccc gtgaggacgc acttccagcc aacggactaa agaagtggct gaaaagccta gtccagtggt ggaactaagc ttttaaaaaa aaagcctctg tttgatgata tgtgaaatgg ggtgggtgat gcttatgatc gaatctgatc aagaaaagga taagattagt gactgaaggg acaggactag gaaggccatt cagatgactt taggtggagc gcagagctgt aggatgggtt accttgcacc gtcctatgcc tcacagctgg ggagtataat tatgtataca tgatttatat agacatggat aaatgcatat gaggtgattt aagagacctt cacgccagaa gccgccttat agcccagccc tcttgcttcc cttcatatct cagctccagg ctagggtcca atcgaatcag tcacataacg ggtgttgtat ccttccaact ccgttctgga cttcccctca tcctttttat tctatcggtg ggggatttgt aaatatttta ttggccttct attagttgca ttgccccaag agggcagatt cttctcctcc tatgtcttta gaacagtggt ccttttgcct cataaaatag gccccccgcc agtaaaagtc tcctgttttt gacagaaatt ttggatctgg ttactgccac tatgacctca tcccacatgt agaataaagt gtttgttaga tctcaagtga cagaggacct gaaagtggct aaatgccagc tttaaaaatt gactgcctgt gtgtgtgtca gaagaccgga ggagctattt cagagatcac ctgctcagtg caccagggtg gggggcccag tttcttgaac tgttcccttt tatctcacca agtctcacct tcctttacaa tatatcccct agttagatga atggggcagc gttggaaacc ggttatgaca agagagctcc gagcaccctc gaaataaatg tgatggaata cattatgttt gtaaaatgtt ttatgaacac caaaaagagg tgttccgttt taaaatgaat aaccaccacc cccctgttcg cattcccttt gcatcatgtt ggctgaatca agggacactt gtgtacacat tttttggctg ccttgtggtg gctcccgggt tcatgtggaa cttaaaccac tccttctcat aataggacat ccggagctcg gtgcgtgacc tacaaaccac 61 gccaccgccg tctagcccag ttaagccatc ggttccagga gcaatgaata gaagagatca tgctttcaat caccctcatg gattctcgtg gaaacctaag aaaagaggaa ggctgagtta acctgcctgc agaaggacag tggaaaagtc tgggaagaga acaagacatc aggcatgagg gactgggaaa cagggcttcc gagccatggg cgagtatcac ccacagccac cttggagtat tttttttttt ctgcattttg gttttatttt tatgtgttag ccctcttcaa gactttggac attcattatg taataccctc gtggatggag ctcacccctt atcggaccac ttgtttataa agacacatac gtgagaccca ccatgaggct tcttggccct caaacctgcg cattacaggc catttaaagt tctgtctgct ctggcagtca aaatgaaaac tccaaggttc tatttcactg tggttatttc gtttattttc tgttgggtct gcttctctcg tctagcgctc tctccccaga tggaccacca gtctcacccc gtatgtcctt gttttgagca tggggcaggt gccttcccaa cacagagaac aagagggtcc tggtctatgg ggggctctgc gaggctggaa ttaaaagcaa gcagggggca ctgtgtggtg ggggttcaga aggctgtgag cttgcaaatt tgattgtgtt agctggttct gacttaaaga tcaacctatc agaccaaggg tgccacctga tgggaggtcg atgctgttta tcctcaaaag gcccgggctg ctggagagcc agtgggtctg atgttgatcg cctatttctc gaagcacata attttatttt tttctgctgt cctccctccc ttcaactttt gcctgaatgt ggtgatagca ccttcctgaa ccaccatgtg gctagcaccc gccacccatt tctgtatttt tccatgttgt gtcctggttt tgccctcgga ggttcccgga tttttattta gatccattca tctagaatgt ctagtctgct ctacactgca ttcaggttgt tgtgtggatg caccttttga cctttgaaaa tcgttgctgc tgaagcacag gggctcagta tcaggggtga gggaagtcca tcactgcccc gtgccacagg aggtaggtgt ttcccaagtc agggttgaaa tcccttgcct ttaccgaacc cattttgtga agtaatacac gagtcttcag ttctaagaac tgggttcaat tggccaagaa tagagaggag caaaatgttg gggggaaagg gcaacgtttt aagcacagta tagaattgat tgtattggaa tgtggctaat gcaggaaaat ggattcttag aaacgaggga tggggtcatt ccccccccaa ttaggcctgt gaagacagaa ccccattagg cctttggaat acatatgtga tttaaaaatt acaataaagt acacccccct gggttgatga ttgtgtttcc ttgggaggtc taggattagt aggacacagt tgatcccaga cggtgatgtt tttttttaat tacaataaac ctccatcgtg gtgcacgagc agtgccagac ataacgtata gtggcagtta ttctatcacc ttctgtctct tggccctttg agcctgtgtc aaacacattc tcactgtgaa tattgtctcc acgtgggctt gctctaggct cttgtggtgc acccatgttc tttgtctcct tgcacccatc tggtgctggg ggaagcccgt tctgggtttg tgaagatcga 561999 62 atgggctaat cacatgggct tcttagccct ccatggcttc ttttatgacg acttcccatc gttgtagggc cagaagggtt ctagaatgcc tccttcttcc ccgtgggggg agaaatcttt ggcatctgtt gattgtagct ccgctaaggg agggctttgt tatttgactt ttcccctacc cagggaagtc ccaggaggac tgttgccttc caagtgtttg ttggattctc ctggttttct cagcttaaga ccaagacagc aattgaaagt ggggtcacag gcctaagaca cagaaaaaga tatagctccc tcctgacaaa ccaggacccc tttgttctag caaagctgtg gctgaaactg gatccattgt agggcaagtg ttttttggcc taccccctgc tgggtggttt gaggtgttgg ttggcttgtg gtctttgcta atcttatttt tggcccagtg gggaactaga aataaagtca aacgcagaat cactaaccag gacctccagg accaggctgc gcaccccact cagtccatgg ttcatgcatt ggacggggga cgacttcact tgttcttgcc tcacagagtc tgagttgtag aggtggcaag gccagagcct gctcttgtcc ttctaatgcc ggtgtctggt gtaaagttct acgcatggga cttgccatgc agcggttgcc atttgtaagc tacacacagt tgtccccagt cagctgtaga gaccccacgt tgtcctgtgg cttcacgtgg ttggccacac aggaatcaca ccctccctgc ctcatctgga tgaggttcca tccgaccttc taggaacttg cagctcccac catgccactt ccccactggc ggggacttcc gttccatccc ggataaataa gtcacaactg ttgccccaca tagtgtttgt gcttgccttt cccggaaetc ggctttgtat tgaccaaaga gaagaaacct tgtttcctgg acacctcaag attggaaggt agaatagaga ccatactctc tccccatccc ctgtggacac gagatcctta gttaagactg tcccacatac tgttcacaat ctcttagttg agactgaacc gaagtctcca ctattgtata ccagtactct ggtcgctaag ggagaaggaa gcctggtagg ttcactcttc tggagaatcc ggacacgact taatgtttat tgctttgagc gggaagagag cttcatcatc tggatttccg tccttccact cacgtatttc agggggaggc cctcccacat atgggtgttg caatgggagt aaactggaga gaccaaggtc ggctcagtgg accctgggct tcaccttgct ccatcccctc tttttttatt gccaggcccc cttttcttat gatcagtaac gctaggcatg ggaccagcag attttcttct aaactaaaat aagacagctg accagaaata ctggtggccc tcattgggga agattaaagt caataaaaaa gcggtcctgg gttcactggg ctccaccttg accctcctgt acggagcgct actcgttcct tcctgctggt gacttctgta gcatgtggga ggagtcttaa tgtcttctct actaaggagc tctggtctct tcatcattgg attacatctg tgtgttccaa agcatggcgc taaaaaaaat cagcatgcag tgggccccct tattcacagg ggttcattgg tgcctggaaa aatcggacat atggcaaccc ctgcagtcca actttcatgc cagggacggg gaatcgactt tttcccgctg tgggcatggc cagacctctt tggaaatcta agtttatatt cctacctccc cttgggatgt aggggggcca ctcctccagc ttttcttttg acagggtcga tagaccccag agctgcaggg gagagttcag attgtctgcg gtgtaattgt tgtatgcctc gggctacact ctgcagtgga ctgtcactga ttgttcacat acttcttggg aactccaggt gaaagtcaat acaggtagag gacgggacgt aggaaaataa actggttatg actaggatcc ttcttaaaaa aaaagaaaga ctgcaaaggc agcaggatat gtttggcacc tcctgggctg gtaaaggctg tgggacctga gacggccatg acacatgatc tcttagttcc ccactggacc ctcagttctg cttgtgtctt gcctttatct atttaaggtc caaagacatc tgcagagggc atgcagccaa taatttggct gatctttggt gcattgggag ttttggggca atcctcataa atcccatgga gactgagcga actccagtgt tggggtcact attggagaag ggagcctggt agcagcagca gagtcatgga caggcaggtc tcttggtccc tccacgtatt cctctcattt caagcagcaa gcagggcctg gagtaatcag acctgatacc catttctaag ggctcagagt aatttggact agaggacaga ccttattcag gtcagcatag catgtgtgtc tgtgtccttg gcgtacatgc agtcttaacc atttaggacc ctccagagac gatactgttc tcaaatccag gtcctctctg cggcctcgga gcttggtagg aataggggca accctgagct cacatgccat aaaaaaaaat aaaggcagta aacttgggtt cttcccgtca aagggggtat agggcctgtt gggctgtttc caaaggaaga ttgtcttctg acagatgtct ctgaccaggg aacagagaga gagaccagaa ttggtgactc cccatggcgt cacccatgta tttttctggg acaggttctg aaaaaaagac gtgccaggtc tgtggcatgt tttgggagtc gaacttcctg cctcttctga cagaggagcc tttcactttc tcttgcctgg aaaagtcgga gaaatggcaa gggctgctgt gcagcagcag ggctcagaga ctatgcctag cgactcctge cttgtctggc tatttattcc acgtcctgga ggacagccag acgtgccttc atctggcctt ttgtgttcaa ttggggggac tggtttaatt ggatactagg gatggggagg gggtcccctc tttatttccg tctctaattt aggatcttag actggactgc caccctcagt acttttccag gacccagaac gccctggctc caattggcag tgtggcatga acggctcttg cagtgaaaaa cccaattgca gcatagcctg tgaaagcata cataaaaagt aatagttcct ggtccaccca cttggagacc tctgtgtgta tctatttact cacaggtccg gaactttctc tttattttac attgaaccca cccatacaga gtctgcagtg caggcactcc ctgcctctgt atccagaatg atttccctag ttccaggtta cttgtctcca tttgttgtgg gggtctagtt ttaaccactg tgcgctaggc gaaggcaatg tggtaggctg acttttcacc agaatcccag cacgactaag cccactccag ctatggggtc caaactcttc ggctgaggca 22344 22404 22464 22524 22584 22644 22704 22764 22824 22884 22944 23004 23064 23124 23184 23244 23304 23364 23424 23484 23544 23604 23664 23724 23784 23844 23904 23964 24024 24084 24144 24204 24264 24324 24384 24444 24504 24564 24624 24684 24744 24804 24864 24924 24984 25044 25104 25164 25224 25284 25344 25404 25464 25524 25584 25644 25704 25764 25824 25884 561999 63 cctgcctgaa gttacacagc tggtgtagac agaacttgga ccagtgcctg agctcctcac 25944 ttttgtccca aggttgcagc ccagcctctc ccagaattca gaccaggttg gagagatgtt 26004 ttgtgtggtc catgctgcta ttaaaaaaaa tgaaactggt cactaaagtt taaagatggg 26064 ccgatgatag atacaaatct agatttctag gcaatggtga cagtttggaa attctacgcg 26124 gcagccattg gccagggtct gtccctgttg gatgaggtct gttccttggt aggcctcatt 26184 cttgccaggt tgatgtatcc tagttacatg gccgtgggga agtgacttgg cccttccagg 2 624 4 cctcaatttc ctcctctgta aaatggggat aatcatagcc ttggcctcat gggttatagc 2 6304 tgtgagggat aagtgggttg ataaatggga agtgagtaag tgctgttggc tgcagttgtg 2 6364 agccgtgagg aggctgcatc aggctggcct tgtagctgtt aattgtatgt gcatgcgtgc 26424 atgctcagtc gtgtctgact ctttgtgacc ccatggactg tagcccacca ggctcctctg 26484 tccatgggat tttcccaggc aagaatacag gagcaggttg ccatatacag gggatcttcc 26544 cgacccacag atcaaacccg tgtctcctgc actggcaggt gaattcttct accactgagc 26604 caccagggaa gcccagctgt taatcaccag cctcttttgg aatacattca gacttagttc 26664 atggaggtcg tccagggtcc tgggtgatgg aagcgttggt tgtagcggga agctgggcag 26724 ttgagagccc ctcagggttc tggcactgct tatggcctta ggtgcattgt aaatctcggt 26784 ttccgtatct ctgacagtac ttgtgetttc tgcctgctta agtgagggaa taatgaaaat 26844 aatttaaaaa tataatttat gactataatt atcattctat tgatagtata tgtggcagta 26904 tatatatagg gagggtgagg attcagaaaa ctggccatac ttgggcagtg atctcatctt 2 6964 cctgagtctc tgtttcctca aatgtcagat gagatgatgc tagtgtccac ggcatggagc 27024 tctctgtggg attcagggag caaagtgctt tcgagtgttc cccaccctta cagagagcca 27084 aggggatggt gggggtggtg gagtgggtgg gaacatggga gtggtgggga tcggggggtg 2714 4 gtggtgggga tgatgatgag ggtggtggtg ctggggatgg gaaagaaggg tgtgtggcag 27204 ggcacatgga atagtgtggc ctggggaagt gggagccgag gtggctgcgg ccacaggtct 27264 gggctggggt tggagctgga ccctgtccct gggagcatcc tggtcctccc tgtcctgtct 27324 tgcaagagga nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27384 tgggacctgg ggaggagttt cgatatggtt ttcagttcct tgagatgcca gtgttcctga 27444 gcaggggaga ccatgatctg atgcgtgtta cagagccctc tggtgctgtg aatagaaagc 27504 gggggttggg tggggggaag ggctggggag tttgggagga ggcagtgtaa ttggaaggag 27564 tgaacagagt ccagatccct tttgcaggga gagtcagcca catgtgctga cagatgagac 27624 ttgcggtgtg ggggcagaag agaagtcaaa ctctttagtt tctatctgag ggactccgtg 27684 aatggtggag ctgttttccc agatggaaat atacaggagg gtggtcaaaa gcaggtggag 27744 ggacttccct ggtggtccag tggctcagat tccatgcccc ccatgcagtg ggcctgggtt 27804 tgatccctgg ttagggaact agatcccacg tactgcaact aagagtcttt gtgtgtgcgt 27864 gcacagtcgc tcagccatgt ccgactctgc caccccattg actgccaccc caccgccagg 27924 ctcctctgtc catcggattt tccaggcaga atactggagt gggttgccat ttcctgctgc 27984 agtatatctt cccgccccag ggattgaacc acgtctcctg cgtctcctgc attggcaggc 2804 4 ggattcttta ccactgagcc acttgggaag ccccacctaa cagtctgtat gccacatcta 28104 aaaaaaagat cctgtgtgcc gcagctaaga tctggtatag ccaaataaat aagtaagtat 28164 atatataaaa ggagatggaa agtatggggc ataaatctgg acttacagtc ttggtaggag 28224 aactcaatca caccaaaaaa gaacccaatt agaaaatggg ccaaggactt agagatttct 2828 4 ccaaggaaga tatacagata accaacaggc ataggaggaa gcgccctgca tcactcatca 2834 4 ttggggaaat gcaaatccta accacagtga gtgatcacct catgcctgat ggaagggctg 28404 ctagtaaaaa aaaaatgaca gtgattggcc aggacgtgga gagattgaag ctcttgtgca 284 64 ctgttggagg gattgtaaaa tggtgtagct actgtggggt aagtctggca gtcagtgcct 28524 caaaaaatga aaacatagaa ttgccatcag atgcaacaat tttacctctg gggtacatat 28584 tcaaaataat tgaaagcagg gacttgaaga gctaggtaca cacccatgtt cattgcagta 28644 ttatttgcaa tagccaaaag gtgaaaacaa tccacgtgtc cctccataga tggatggata 28704 aacagaatgt ggttcatcca tattttggaa tgggattcag cctcagaaag gaaggaaatt 28764 ctgacgcagt ctacaatatg gatgaacttt gaggatattg tgctgagtga aataagccag 28824 tcatgaaaag acacatattg tacgattgat tctacttatg ggaggtcccc atgggaggca 28884 gactcataga gacaaagggt accaggcgct gatggggata tagtttcagt tggggaagat 2894 4 gagaagttgc acaatgatgt aaatgtactt catgctgctg agctgtagac ttaaaaatgg 29004 tgaagatggt acattcgatg ttaatgtgta ctggagcaca attttaaaaa gtcatgatag 29064 agtttaacct gagagagggt tggtacctgt ccccttcctg ccccccaacc tcatcccggt 29124 ggcatggatc gggttcagta ggtggtagac cgggttgccc ggcccccagg gtgattacag 29184 cattcccagc tgcttgcaaa agatgaagtc tttgaaggag gcaaccctga gccgctgatt 29244 cttccaggct tacagcacca ttgcgccatc ttacaagacc ttggtgagtt tccccatgcg 29304 ttcttgaacc cgggccaggg tgagtggagg aagcctcatt tagcaaccat cgttgtgtgg 29364 cctttctaca cccgggtgca ttcttcagag cagtgcatgt ccttggtcac ttgattcttg 29424 gcgctgacac cgaggaggaa ggggaggtac tcacgtctcc atggcgactg agggggaggg 29484 561999 64 gacaggggtt ccaaacaggt gacatccctg cgtgagtttc caaacattta gtatgactga ctccaatata tgcccagaaa catgcttgag ctatcctctg tgcttgtctt tcggtggagg tgtttcagca ttaccaaggc tctgtcacta cagttttctc taaatgaaag caagtcgctg gatgtttcca gactttctga tgtcacacac ttcttcctct tgggcctgta tggaagcaag tacccagtaa tcaagtaacc ccaagggtga tttatcaaaa aggcttgtga accattgtgt gctaccagga cgaataccca ttccccagta tttagtagct tctttggcca actccctgta gccagtgtta ttgtgtgtct ctgatgaact ggcagagttg acaaactcag tgaggtctta cagctgctcc cctcccggtt atcaacaaat ctgacagttt ggttgcaaat aaaaagagca gccttcctct ttcatcttag gcttgttaaa gacggacctg cacactttga cccgtgcctt tgaggaattt tttgggtccc gccaggaagc cccaatctgt tccccaattc tggctgacag accacattag tctgtctcag agtgggggct atgtaaattt agaatgtatg atcctttggc aaatagaaag caggcaccag aaattgccca cctgacttat aaagagcagg atgagattgt gtcttctccc agtagaatgt ctgggtttgt atcaacaaaa cattgcttca gtataatagg gatttattca gccttgccct atcactcaaa tattgcaaga ctacagtcgg tcaggatttg atgctggcat agcagcatct ctgctaccct tggaatcaca gatagaccca gaatatacag gtagaactgc ggaatgggat gtatcccaga gattccccaa catcacgtgg gtggaagtgt aatgatgttc gaatcatttg ttgcttcccc gaatgaatga atccctggaa gggagccatg ttagctcagc ttttagaaga tcaagtattt gtggagttgg tttaactggt cagcaagatt ttcatctgta tctctgacac gcacagatat agaatcagga gaaccactgc tgaagctgcc tccaaatgat ttttgcaggg ttcctgagac cctgtgggaa tgatgctgcc tgcagctcgt tggtagagga cctctcacct tgtctaagtg catgtgttgc gttgggagga tgtccacctg ttaaaaaata gccagatgtg ctttcttatt gtggagaaag gccagttgca gcctgattcc tcccctgtga tagggcccag gcccttgggg cagagcagat gatggaatcc gagctttatt gcagttcagt cataatctca gccagaaaga ggattttttt gggccatctt ttttgtggga catcatatca tagaatcccc gacttcttcc cagtgggtac cattgcatgt tgatgtcctg tgggagcatt tactggggtg gtgtatacca cagcatgcca catgcgggtt ggagtcctaa cagtgccagt tgaataacct atgacacttg ataccatatt gggccatgct gagcctgtgg atgctaggct agctggaaat tgaagtacag cttcaggaag ttaaaagaat gccacgtgcc ccgcaagtat tggttctcta ctgagtccct tttctgtcag tcaaacaggc accaggctgt gaactttttc ctgagggact ccttagaacc cctgttggct ctggggagag ggagttcttt caaatagtaa ccacatggtc ccacaacagc caataagtat gagtttgggg aaattatcac aaaatgtaaa gcccacaggc ggggtgaaat tggggctgaa ctgcctggag cctggggtca tcctttccca gctctgggtg gcaagtaagt aatggtacct tttgggctga attgtgcttc tatttcaggg tgatgactgc gggatggtac tttttttttc ctctgagatt gaaggggata gacaggcaga tggtgctccc agcatagctt tgatattgtc ggctgttctt gttaataatt tgtgtggacg ttgggcatgg gagtgtatca gcctattgac cttagttcct ccgctgggcc agatatgctt gtcattttcc aggaccagtt ctcatcctgt gacaatgcca tgattcggat acgtggggat ccagaacgta tgtgatccca acagatctgt gtgttatgaa cttcgcctgc ttttattctg actgtctcat tccccagaga gttcccaggt gcatggtaaa tattccgtca agctgtttag gtggctcagt ctgagtctgt tttggagggt gggaggctgg ggggtgtgtc atatttttca ctctatgtgg tttgcttgag tcttgtttct gagaatggat aacattgtta accattgtta tgtagtttgc tggaatgttt gtcagtcctg ttcccatcat tagaatcgga tccacaccta cagactagag ggtttagtct gcctcatgga agcaacagaa gggagagact cactgggtca catagtgcca aacgggagag tcagaagcta taaggctttg aaggtgacct actttcacaa cactcctttg agttttacct tggcttcttt cctcactgct gggttgtttc tggattttca acgtttgctt tcactcatac agtcctgcca caaccagtga gccagggaag aatgagctga taatccctgc gtgagcatag gttattgtag gtgagtaaag tgcaagtcct gcagacccag cctgccattt aactcatcca ttctctgggg aatttcaaat tttaatagac aagcaagtat gtggcaggag tgctgagtcg gatgttgctg caatgatgta aaaagaattc aaatgtcctg ggtcacttgg ctgtcctctg tgctttggga cttttaggcc tcagttcctg actttagagg acatccttga gacaccaact tttttttctt acatgtacat atcggctgta gctcccagga tgaattgttt gaaggaaggg ggggaaattg ggtggtgtag actgcgtgtg cgatgagcct ttgaggaccc ctgggtgcct ctgtcagatt aacctgcttc ggaggtggtg gtgtccatgt gacatcacac ctctgttcag gacaggctca tgcccaatac ttgagcctgg ctgaacatcc ccacccagcc ggttttgtac gctcaacatt gtgtaaatat tggtttggag tttctcttag tagtagctga cagagtttgc atgttttgtt tcgaaactgt tcccactcct gctcttccta tcagctctgt tagatgctta gtcagggctt cagcctggtg gtgccaggag agccagcaca ttaagatgtt gtggccaaat gtgcggtagg gtgcatttaa atcgagtccc ctgacatcag tcccaggagg gcagatttga gtccagggac aagcaaagga caaggtatag ggcccccacc gccctgcccg acctttgctc gatacccccc ctcagctttc ttccaatgga 29544 29604 29664 29724 29784 29844 29904 29964 30024 30084 30144 30204 30264 30324 30384 30444 30504 30564 30624 30684 30744 30804 30864 30924 30984 31044 31104 31164 31224 31284 31344 31404 31464 31524 31584 31644 31704 31764 31824 31884 31944 32004 32064 32124 32184 32244 32304 32364 32424 32484 32544 32604 32664 32724 32784 32844 32904 32964 33024 33084 561999 65 caccctgtgt ggcctgtctg ccccaccccg tgtttctttt cacgggcttt tgttgtggag tggtttcggc gctctgcggc aggcagattc aagttcacac ccaccttgtc aggggcgcat cggggctgtg ccctcaaaga gctttgtaga gatgggacta tgtgatgatg ggaactggaa gcagacacct ttcgactgtg caggcaagaa gatcgaaccc cacctgggac ttttaagcca tttgcttcca ataaattctg tattattatt tgcacacagg ttgcattgca atatatctac ctggggctcg tgaccctcat tacagaaaaa tattgttaac gtcagcactt aaacaattgt cgaaactgcg ccttcatctc tgtgtttcct ctggatgatt gtctgatgtt gctcagtgct ctgtttccaa agaagggcag ttgcttgtag tggtctccca tgttgagctt caccctttat cctggcagtc tggtcctgaa tagatttagc tggatgtgtg gtcatcccaa tggaatatca atctgtgtcc tcactcattt gtactgactg ctgacatctg tgagtcgaat aagtcatggg cattgtcagg aggccccaaa ccccactgca tttaataatt cctctacttg ttcaggttct tcacgggctc acgtgggaat tttaccactg aaggaggggc ctggcggggt ggggaacaaa tgggaggaag agttctaatc tgtgattaca acgtcatcat aaagctgagg aagctaggtg tgattttagc cgaccccata tactgaagtg atgtctctta ctatttttga gtacatgttt ttaaagctag gtttgggttt atttttatgc cttagtttct aggtggtttc ttttaaacat cgtttttcct ggagccctgt tggaagagac atctttaatt tgttggggtt ttttggctgc ccccctgcag cttttttccc taggctctct ttgaagagca cctcttgtga gtgctcatct accctgttta gaagttagga ggagggcctc gaggcagcct ttgtgggctg attaaaattt cagtggttag actaagatcc atcagtgcag tttggcacgc ggtgctgcaa catcagtggg attccttttc gtggacatgt taccaaactg tcggtctttt ggacctttct attgtgtttc ccaggctgtg ggggcttctc gcacttgcaa ttatttatct tggtgagtgg agggcacctg cggagcgcag ctcccagatc agccaccagg actttaggag ccctgcttgg ggtcgccccg cctcacatgt ccaggacctg ttaaggagtt aagggtcctt tcggagtgat actggattct tctgttatgg gactatagcc ggttgccatt ggtctcccac tttctgacct gataatttgt ggaagtaaaa aaataaaata tgcgtcttcg ccatggcatg ttaaccggtg ttcactattt ttgcttctca gtgtcttgat aatacggagg agtgtagtcc tccttagttc acctcgaggc tggaagcata tcctaggatc tggctgtgcc ctagttaggg ttagattgtg cattatgtca caggtgagga gatggggagt cagacccccc gagagcttca aggtgcctct cttttgcttt ggctctatac tgcaagctga gaaagtgtac tcacctgtgg cccccgcctc cgtcgagttt tttgcttggg gtgttattta cactttcacc tctcgttagc ctgaggactc ctatctttct ttttggtctt gcagacctga gaaaatccac atttttggct ggatcactct ggcttcagta gctcagtagt aggggttgaa gaaccctccc gacagtgggc tggctgcggg ccccggcagt ctgagtgggt tggatgcgtt cagggtggga gtaagaggga aagggggcca cctagaaagc tgtgtgcgtt ctctaggctc tccttctcca actggcagat ccaaagccgt tacatggcaa aaaaaaagaa tttaaactta ttgtggtgtg taagatcttc gaccaccagg tctcaatgaa gctctgatat agcttcctat gcgccctcgt attagtggtg ctatctcatc atgtgggatc gggtcttaac ccgtccagac tgtttctcag gtttggtcgg gttctgggtt atatgactcc aaccagggca ggctgactgt ttccttctgg gaggtcagat tgagggagtg aatttttttt ttccactgca gtggcgaggc aaccttaggt aaggctgtcc tagcaccaga ctgttggtca tgatgtccca gcttcaggag agtgtgcatt ccttttggta actgcctagg tctttaccat agaatttggg gttttgagaa ccagactttg atgctgggtc ttccctgcgg gttggggcat tgtggtgcac tctgtgtctc ccagtctgtt ctcccgcagt caaggatgcc ggggtgctcc atgggaagca acccccatgt agattgtccg ggcaggggag aggaatgcag ctccaggagg gttaagtcac ctctgtccat ggggatcttc gggctcttta tagatagtaa tagaaattga aagccagggg atgttttatt tgggctttct gctccctgat gaagtcccaa tttactgttc aactcagcca ttttaaaaca ttcccacact atccattagt tcctttatct ttagtttcct cgctggacct accatgccac gtgttgttgg atgcctttcc tgggcaggaa ccagttgagc cagaaagttc agacatagag gtctccagtg ttgacaggat ctaaacagct cattaaaaaa gggcacatga tgaaaaaaaa gtagaggtcg tgaatccctc gactaccgtt tcgatgtgct ctgtatgaac acactgccac ctggtggctc tatggtatag gatgttgggg cctttgtgtg aggaggaagg caaaggcact ttctgccccg ttcgttgttg tgacttctcc gtgggttcag gggcttacat ctgcattggc tcttttaagg gggagctggt etggaaagga tgggtgtgga ggatgatggc gacaaaaggg gaattatcta tcagaaagga gcggattcta aaccagccct ttcagttatg gggaattctc ccaacccagg ccacgagtgc atgtgtgaca atgcaatgaa taaatttata tatttattat ctagttgccg cagggctccc tattgtttta tggagcatta ggcactggtg gttttagatt tggcttcccc ataaactgtt tttcccttta ggccaaggat ccagagaagt acttagtcgt ttttggtgac atggggattt gagcacagag ttggtcctcc tggtagatgg ctgggtcccc gtctccttgc acagtctccc caaaagccct atctaaattc gtttgatccc aaaaaaattc aggaattacg catgtcagct gcccattccc cgtttaggtc atgccatttg atcattttct cacatccttg gatctctttg tgactccatg tggcttccat 33144 33204 33264 33324 33384 33444 33504 33564 33624 33664 33744 33804 33864 33924 33984 34044 34104 34164 34224 34284 34344 34404 34464 34524 34584 34644 34704 34764 34824 34884 34944 35004 35064 35124 35184 35244 35304 35364 35424 35484 35544 35604 35664 35724 35784 35844 35904 35964 36024 36084 36144 36204 36264 36324 36384 36444 36504 36564 36624 36684 561999 66 tctcaaggct ccaggcagta ctgggcagac atagtctctt catgcagtga gacttgagaa gcaaacggtc aggaacagtg gcgattagtg ttgggattta atgatcagat aaaggcaaac aggggccagg gcagccaaga gactcaactt tgtttttttt ttagtcaagg tgagttgagc gaaggaggcg tgagcaaact accctacaac cctgggatcc gaacccggcc tcctgttacc attaatttgt tacaggcctg aggtgatatt caatgaggca ggattctctg ctttattttt aagatcttcc cacagacttc gttccctgac tggtccacca tggaatgggg cttaggtagt attggcagtc gtggttcaat ctgagcaggg ccattactgg tggatcagat ggagggggat cctgagacca agcccacacc gtcatccaga caaggggagg ccccttgaga tcccaggggt tctctactcc ttggagggac aagcagaaga gcaccccctc ggtggaccac tggccagtga cattcagttg ctttcaccat accatctcgt tcttttccaa cataagtcct ttacttaatt gcctcatgac ggaaggggga aaaacaggtg tgtccatttc gaaaccagca atgaagggat aatgcataag aggccaatgt tagctgcagg atccctagat atacctttta atgagagaac ggtggtgttg cttcctgatg cttagcttaa ttttttttct gtatggcatt caactcattg acagaggacg ctgggagatt tgaacaatga aggaggtaga tcgtacaggt ggtgggggca gaaactcctg catgttgatg tggccctcag aggtagacgg gcttccctgc taaaaacatt ttgcgtcatg agtagttgcg cagggatcga aggaagtccc tgactgtcca tccctgaagg agagtcatcc caacaccctg tctccccctg cgaatgtgat ggaccctaag gcgatgatgg ccaggggctg tcaattccgg gtgtggcttt tttcctggtg gatgctttcc gaggacagag aagctcatcc agttacgtga ctggtctggg taggcagggt tgctgatctg agttgcagat tgtctgactc ctcccagagt cctctgtcat tgagtctgtt tccagtgaat gagagatttc ccagggtggc cttgtcaaca atggatggga atttactact aatattgttg cacctatgtg ccctatgcct ggctggatca gagccagagg aagtagggag aaccagtagc cagtgaagag gaaaaaagtg gatggttttt gcaactcatt ttaattcttc cccaaatcca gaaaagaccc agatggttag ggagtgccgc caacaaaatg gagtgactgg gaatcagagt ggattgcgtt tcctgtgtgc caaaaccagg gcctcagtgt aagagttttg ccttccccag tacttattta tgggatcttt gcacgaaggg aacggcatcc ttgttcatct tggaatgacc cttcctggag tgggagcata atcactcagc ccccaccccc cttatttgga tccaccagga aggcagagat gaggagcaag acttctagcc gttacagcag gctggtttaa tggcgtctgt gcccctggag caggcttgtg agcctgggcc gtcccacttc ctttactgcc agtctcagca cacattttaa tttgcaaccc ttgctcaaac accattctcc cttcgtatca attcagggtt tctagtggtc tgctgtagct aatacatagg tgtgatgggg atagtcccca atggaaggcc gaccttggag accaggccca gagtgagcaa gtgtgagact ctgtgggagg tctgatctgt atgactgcat gatggatttt gggtgggaga ctgctgtttt ttgcctttaa gactgaatca tgatgctggg atggcatcac agtccatggg agccctatgg ctggagcggg cttggctcat ttgcaattca tccgagccca ggctcagtct ctgtgtctgt ttacgaccag cttctcaggg tggctgtttt tgttgcaatg cttaactgcc tctgtcttgc atctgtaaat caggtcttaa gaggtggcac gaagagtgca tttgccctgg ccaaaaaaaa aatagggtct atgtctttat tggagggagg ttaggctcat tctagaggtg cccaaggagc ccgggctcgc gagcagagat ttgtggggcc ctggaggcct tttcccctgc tctgcccctg ccacccagaa tccattcacc ttaaaattat catggactgc tcatgtccat tcctgccctc ggtgccaaag gatttccttt cagtggttga caatctatca aattgctgat gaaggactgc ggacctagaa tcattgagaa agaagattgc gaatcttggc ggtggactgt ttgtaggtca tgtttgagca gtggggaaca tggtctaagc gggtgtgtct gaagtggagg agcaacgtgc aatcaaagga gcccacacta agagattgag tgactcaatg gtcgcaaaga ggctgggggt aagagtctgg ctggttatta ttgggcagat gtgggagaga cttcctgcac tcaatgggtc tgctgtacag accccatctt gggtcttagt cacagactct gctcggcatg gagcaagaat tcagaatata ttagatcaga ctggagagtg acgggaggca ctttttctcc gtatgtccaa ttgcagatgt aggaagagga tggctacaag cccctggaac agagaagata cttaccgctc agacttcctg cctgtgatct cctctcagcc gcctggctgg cttctgtggg ggcctcagga aggaaatggg ccagtctcaa aatgtttgct agcacagtgg tgagtcggtg aatctttccc tattggagct agcattgtaa gactgcactt tgactgcatt gttgataatg tttagaaagg aagagcctga ggtgacttta ccttggagga tggtgggctc aaagggagct ctgttagagg ggaagatgcc gcctagggac aaatgatgct tagagcaaga agtcaagcat ttgggtttcc agtcagaact cagtcttccc ggcaggagga gacatgagtt gtaggatacg gtctctggat tctcgtccca gaggtgtgtt cagattaatg aacatgagga ctgtgacctc tttgagaata atggtggata attcatttat tgcggcatgc agttgtggta tgggatctta tcttagccat gcatctagca gcaggctcat gggttttgga gtaggcactt ctggattggg gtcctaactc gatgaaagta cacacagaga ccaaggaatg cttcagaagg gatgttttaa tggagatggg gccccagtga ggctgctgac tctgcttgct cctgagttcc cctctctgtg catctgctcc agctgttgga ggctgcggcc gttggtcagt gctttcctgt atgccatcca agcatcaggg ttagcttcag tatttaagat ccactgctga 3 6744 36804 36864 36924 36984 37044 37104 37164 37224 37284 37344 37404 37464 37524 37584 37644 37704 37764 37824 37884 37944 38004 38064 38124 38184 38244 38304 38364 38424 38484 38544 38604 38664 38724 38784 38844 38904 38964 39024 39084 39144 39204 39264 39324 39384 39444 39504 39564 39624 39684 39744 39804 39864 39924 39984 40044 40104 40164 40224 40284 561999 67 gggcctgggt ccctgccccc aggagaaggc tcatattgct tgaataaagc gaagaacagt catagageag gaggcatgtg aaacattgag tgtacaaggg gtcggatctg gcaacttaaa agttgttgtt ctgtccttca tgaacaggcc gtctgccaca aagcatggaa atcttgtacg cctatcttgg gacgaggaga ggaaaggcgg tcctgggcgg cgaaggcggg gggcttgttt aatttgcagt tgcagttggt aagatccctt cagctggtaa gataccctgg gatcccaaag cagttaataa gacattcaca cccaaaagga agactgtttt ttcagttcag ttcaccaact cacctcatcc ttttccaatg attagtcctt cttgcattcc tatttttttt aattgaaccc ctcccttgcc actgtcttat attccctttt atctgtcagt gagcacagaa ttgttgccag gttggccaag ctaggtccta gcaactgaga taaaaagatt gtcttttgta gtatgattct gcaggagacg cctgcagaat tctctcaaga ctattccgtt aaaagcatat ttttgcacag tcaatccctg aacccctact agatagctgc gagtgcctgc aactgaggat agacataatt gttagggggt aggaaagatt ggcagagaga atggcaagga gagaggccac taaaatgttt cagtcactca ccatctccca atagtgactg aagcatgtgg ttttaaccac catgtgggaa gatgcgagat gaatatctga gagttcgttc ggtcagcagt ggcggtgtgc ttctcaggtg tagtcttttt aaagaatctg ggagaagggc agaatcctcc agaagggaaa agttggacac aaaactcacc atgttgtata aaccctgtag ctgtgcctat ttcgactctt cccggaactt tctgttgtcc agtcagttct ccaatgaata aagggactct tttttttaac aggccacagc tgttctagac tgcctctagt cttcctaagg ggacacttgg aggctcatac acaggtgagg ataccttgct cgtgccacac ctggagcaaa aatgatcagg tctctgcttg ctttacaggg taaaagatgc cccatggaca gtctcctcta gtatagatac ttgggattgg gctgtgtccc atcagggaac ttgtagtaec tccctagaca tgggtgccaa ctggtcctgg aattataagt tcagggattt aagaaggatg acagcctatg ggccagcatg agggaccagg catggtattt gtcgtgtccg gagtttgctc ttatctttct gatcttagtt tagaccacca tctgtagggc gactggcgtg gtagcggata ctgaagcttg gtctgaaagc tcacattctt acttaatttg tttttctttt cctgcaatgc taaggtaccc tgcagtgtgg ggctactcac aactgagcaa atttaaacca accattaccc ctattaagga cagttcagtt ttcgacccca gctcaaactc ccttctcctc tcacatcagg ttcaggcttg ccagaatctt catcttgcac cctgaaagcc acttcacaca aatgttttaa ctgaataacc gtcacatcca gcatcctttt tatgtgtatt ctcattgcag ctaagagttc cacattaaga acctcaaaaa ttttcaagaa cttcgcaggt aggttcagtc gaggtgcctg gcaccataat acagagttgg tggactttaa ctgccagtaa taagatactg ttaagtacaa tttctactct cctctgttcc tggctttatg atgccagaag gagggtagat agagatttgg cagaggtcct gctggctgag ccggttggct tgcgtgtttg actctgcgac agactcatat gcagcttgtc ccccaaccag ggcaagtcct acagttgtcc agtgtcgacc atcctttggg tttccgtcac cagtgtgcca gggcatctct tgtccatctt tttaaagttg aggagaccct acccagtatt gagacctggg tccagcattc ttttcacttt ttaaaaacca acatgtctag gtcactcccc cagttcagtt tggaccgcag atgtccatgg ctgccttcaa tggcccaagt atttccttta cttctataga agcttatggg tggaatccta aatggaatcg aagttccctt ttggctacag ccttttggct aagcaaggcg tgataaaagt ggggcctggg gcgtgccgca caacaacaac tattttgcta atagagaatt cactctagtg cctgcatggc gagggctaca ttgaaagcat tttatccatc ggtagatcac caccctcctc caaacctcgt gctttttaaa gattctgttt aggcctggga atctaacagg gtaacaatga agggtgtgtc ctatgctgct ggggcagggc taacagggac tctaggccaa aaaaattcat cccatggact ccattgagtc tttttttcac gaatcgaacc tcagtgattg agctctgggc agcactgtaa agtgtaagta ccaatgtgtg agcagggcac gtctccccac ttgggaagtg tgtcaaggct ggttctattc cttggacttc tttgatccca tggcctggag cactttcttt tacgattgag ttccagaata atgctcccct ttctgcgcct catgccaggc agtcggtaat tctttcccaa attggagttt ggatggactg tctgtcccta atctcagttt accactagcc tataatacgt atgatgtagc cacattacat cttatagata tgatgaaaat caacaaactg tttgatccct cctaaaagat agagtcagca agtaaaagga atgggtacat gtaaagaacc aacccactcc gtccataggg cggttctcct gtgaacattt gttcttagaa gtcttttttt agtgtgccac acatacacaa gttcctgagc gatccatcag agagacagat ctctggaaaa ttcctgagag acctcatgag tgtgcctact acggcaggat cactgtgctg gtgaataggc gcaggcttcc ggtgattcca tcaggtttct catacattag tttttcagcc tgcatgcgat gaacaaaaca gcattgtgct caggcagtga cgtagagtca accttttcct atcttgtttt tccctgattt ctgggttggg ccttgtggct gagttgggaa tagtccatgg tttcaagttt tgacatttag ttttcatcac ggcaactccc atcagttcag tcccctgttc ggcatccaac catcagggtc cagcttcagc gtttgatctc tagatttgcc cctgacttgg tgccagggaa gtttttttct acagatgaga tcatccattc atgctgccag gtcccatttc ggacttcctc ggtcagggca cacccatgct aactacgctt gacgtttatg ttgaagactt tgcctgccaa agtgttcttg ttgcaaagag ttttatctat gagttgtttt gccacctagc catgacaaag 40344 4 04 04 40464 40524 40584 40644 40704 40764 40824 40884 40944 41004 41064 41124 41184 41244 41304 41364 41424 41484 41544 41604 41664 41724 41784 41844 41904 41964 42024 42084 42144 42204 42264 42324 42384 42444 42504 42564 42624 42684 42744 42804 42864 42924 42984 43044 43104 43164 43224 43284 43344 43404 43464 43524 43584 43644 43704 43764 43824 43884 561999 68 cccaactctt gctcagagaa ttctcttggt gaatcagcta tgcctggtcc agcggtgcaa ggtcactttg cctgttctaa tatcattgca ccataaggca gagccccaga attcattgat agcacagcag ttgcttaaaa tttaagccac acaacttccc gtggggttca gtattcttgc tgcaaagaat gaagtgaagc tccttccttt accgtggact actcctgtcc gctcctgcct ataatattat ccacatggta gggcccaggt aaacccactg acaaacatgt gatgagggga cttcgtatta tgaggtcgga gcaatgcagg gaggaaatgg ggcgggcccc attcaccaag gccgggcccc gggtgaggtg gatgcgccat aatcgggaga tggaaaaacc gttgaaggtg gacaatgatg tgtgtttttt ttccagtgta taatatatac tgagtctgtt gatatcatag agtttgatcc attgtatata ccatgtcttg aatgagtttt tttttagttt tccaccaaga atttgtgtgt tgctcaggtt tgccagtaca cagctgagcc gctgcaacta aagactcaac tctttcacat tctggttgga ctttgtgtgt tgttgggcaa taatgcttct agtccaaccc tgccaggtga gcattttaca cccattttat aagctggctc accccacagc tcgaccttga aattccggaa acagggctgg tttactgctg taatggctca atccctgggt ctgggaaatc cgtacacgac catcaccaca ttatttatgg gcagcacgcc attgagtcga tcaatctttc tattagtcat gggagctccc ttggtccctg cattgtatat aaaaagaagg tggagagagg aaatttgaag attccacttt agacacagaa caacccactc agtccatggg cccgtgctga agcgtccctg ctctggcaag ctgcctcccg tgccatagaa tgagccccag agggccatga tgacgagcct tttttttaat cagcacagca ctgtccccac tctgtcttac gatatttgtc acattgccac tgccataact gctattgtaa gtccagatgt tttgaggaat ttgtaggagg tgctttgtac ataaaaatga ggggatgtgg tgtggaccac gaaagtagcc gcagccaaag gccctctctt taagagggag gggctgtgat aaggccgtga ggtccagtgt ccaccagagg ctgcttggct aaggcattta ggatgaggaa atgggagatt cccttctgtt tttatccctg tttcaaaggc gggagggagg tatgattaac gcgggtaagg caggaagagg ccatggacag tgagcgacta atctacgttt ttgttgttca aggcttccct tgatgccatc gcagcatcag gatgaataaa tggcggtcca gttggggaac ttaaatattt aaactgggtt gcacgacaga gtttccatcc cttggcttcc gatgcaggct cagtactctt gttgcaaaga gggggcgggt cctgtgacac tgtgacaaag ggtgtgttga acatctggat ggacctcggg ccctcttggt agaacagcac tttgtattgg atttgtatgc ccttcctccc aaataagttc tttctttgtc aaatggcatt tctttatcca atagtgctgc atgcccggga ttcaatactg gttccttttt aaaccacatt tcctggattt gtttgatccc aaatactgag cctgctccct ataaataaat ccaggcagcc ctgcgtccag ctttgacctt gcttggcctg gacggaaaga tggggcagca gacctgctgg aacctcacac actgaggcac ccctgtgagt gaagagagaa aattaggtgg ttctccaggc caggaactgg ataataaatt aatccgcctg ccctggagaa aagagcctgg agcatgtata aaacatgtcc gtcgctaaat gtccttcact caaccatctc ggtcttttac agatctgtat gtggttagga tatggtctga gctaagaggt gatgtgtttt ctgcctccgc gacccccaaa tcagtggctc cgatctctgg gcctgggaaa gtcggacatg ggcggggctg ggagaggtcc gggtctcctg ttttgaataa ttctggcagc gcccagaagg tcgaggggtc ctgacacaca tataaagttg agtagtgtct tggcacctat atttgtatca tgacttactt atttcatctt ttcatctgtg agtgaacatt gtgggattgc ttctccatag ctccacaccc ttctcgtcac ccctggtggt tggtctggga cctgcgtgct gcaactagag aaattttaaa ctcttgcctg gaagggatca ggggggtggg cgcactgtta aagttatccc atgggggagg gtccctaggc cagacctatg agaaaggatt gagtacccac acgtaagcct gaagaccagg ttctctgggt aaactgtttt agttgtatgt caatgcagga agaaatggca cgggctgtag caaccggatg atcacctctg tgtgtctgtc atctcccgca atcctctgtc aattatattt agtgcccaga ttctgtgctc caagcacagc tagagcttat cttetgaccg ctgtgggcct cccaccaaca aagtggtaga gtcaggaaga tcgcacggac acggggcaag gagaggagcc tccgctctgg gggtccagcc taccgtgttg tcacataatt gagttgcagc atggtgttgg ggcaatccct atttacagtg gtgtgttaat aagcttgttt tattttaaga cacttagtgt ttctatggtg agtagacact ggggtgcatg aggatcatat tgactgcacc tcttcagcaa ctttatttgc tcagcggtta agagtctaca acaactactg aaagtctgca aaccttaaaa tccagaatga ccttggcttt ggcagggata tctaatcagc tctgattgta gggaagggct tctacttgcc agatggatgc aaaaacgtat gaacggagtg tcatctggtg agattaggaa cactgtacct ttgttttaat atataattat gacacaagac acccactcca tgcatggggt agtttacaga aaagtttcct tctttgcaac gtttgctcaa gtccccttct ccaacattat gttagcagtg tcactgccca aaaaaataga gtgttttatt ctgttctgtg gtctttgttc acttccaggc gaaactgcct tcccctagag agaggagcct cattcacctc acacagcctc gagatggtct gtggcctgag ctcttgaaac tgggaggagc tgctgccttt ttggggaggt gggaaatgct ttatgtgagt cccaacctcc tctgtgtcta ttcctcatgt gacaatctct agaatattcc taggtaattt tatcttttcg ggtaatttta agtttacatc ggaaacgctc atgtactcga agaatatgcc tgctgtgggg aaatctgacc tgcaacagtg aaaaatgatc 43944 44004 44064 44124 44184 44244 44304 44364 44424 44484 44544 44604 44664 44724 44784 44844 44904 44964 45024 45084 45144 45204 45264 45324 45384 45444 45504 45564 45624 45684 45744 45804 45864 45924 45984 46044 46104 46164 46224 46284 46344 46404 46464 46524 46584 46644 46704 46764 46824 46884 46944 47004 47064 47124 47184 47244 47304 47364 47424 47484 561999 69 cttgtgactc ttgtagctgg taggaagatg tgttcatcaa taaaggattg ttacacecta 47544 aggcaagtct ctgatctgat attttcaaga gaacttcgaa atccaggttt tatgttaaat 47604 catttttttt gttgttgttc ttgacaatga gttcaacttt tcttcattga agtgccatgt 47 664 agatcaaatg aagcacagtt gaggttacag agtcactggc cccattgcac agatgggaaa 47724 actgaggttc agggctggcc aacttgccct caggtctgac tctctccaaa tccacttctt 47784 cccaaaatct accagttagg gacatggggc tgggcctgac tggccagctc cagtcttctt 47844 ccttctccag agcccctgcc caccggttct gggacagtct ggggacagtg ggagtggctg 47 904 tccttgtcct gggaggccac attcaaagaa aaagtgaagg tgacgcaagc tgttccagac 47 964 acttggcttt gcaacatgga gcaactacct gatgggctgg tcaggttggg agcggggtta 48024 ggtgacaagg ggtttttgcc aagctttata aacctggaga tttatagttc atatggcgct 4 8084 agtgataaag aacctgcctg ccagtgcagc agacacagaa gacacggatt tgactcctgg 48144 gtcggggaag atcccctgga ggaggaaatg acaacccact ccagtattct tgcctggaga 4 8204 atcccatgga cagaggagcc tggcaggcta cagttcatag ggtcacagag tcagacatga 48264 ccaaagcaac ttagcacaag agcacagcac ataaatttct gtatttctgg cttttcatcg 48324 aaaattgctc ttgttctcat ctcctgccct cccactcaca ggctctgctc cccaccacac 48384 tggcctcctg gctgttcccc attgctctgt ctcagttctc ccagccccag ggcctttgca 48444 caggctgtat cttatgcatg aaattaccca ccaccactaa atccctccag tgcctttagt 4 8504 ttttaatttt ggtttagttt tttttgcttt gtttttcctt acattttcac ctgcctggct 48564 cctctgttta aaattgcagt cctcctccca gtccctgctg tgttttcact atgtcctttc 48624 atacaccaca tgttgtaccc taattgttga tgacctgtct ttcctctact ggaataaaag 48684 ccccgtgaag ttagagatct gggtttgtct tgttcgcttt tctaacacca gctgttaaaa 48744 gagggtctgg catgtcgtag gtgttcagta aatgtgtatt gacatggaga acaaacttag 4 8804 ggttaccaaa ggggaagggg ggagggataa attaggaatt aacagataca cacaactata 4 88 64 tataagtaga taaacaacaa ggacttacta tatagcacaa ggaactatat tcaatatctt 48924 ataataacct atagtggaaa agaatctgaa aaagaatata tatatatata aaattgaata 48984 ttttgctgta cacctgaaac attgtaaatc aactatactt cagtttttca aaatgtgtgt 4 9044 taaatgaatg agtgaatgaa tgaatgatga atttgacaca cggagctcag ttcccaccag 4 9104 gtgtcatgag ctgtggttga gcagtggcca cctcccagtc tcttgatcag gctgttgtta 4 9164 ttataacaca tttaaaaata atgcatacaa ataatatcaa ctattaacat taattatgtc 49224 atatgtcaac tctggagcag catgtgtcaa cagctttccc acctcctcct gctcctagaa 49284 catctccagg gagccccttc ctcaatgacc ccgtggagtc tgtgatgcag gctggcacca 49344 agcagccctt ttggagcctt gtggtagggc aggtgggtgg gtgggggtcc tggccctctt 49404 cctcatgagc ttgtttgtct cgtcccccac ag aat gtg cgc ate gac ccc aac 49457 Asn Val Arg lie Asp Pro Asn 45 age ctg tct ttc aac atg tgg aag gaa ate cct gtc ccc ttc tac ctc Ser Leu Ser Phe Asn Met Trp Lys Glu lie Pro Val Pro Phe Tyr Leu 50 55 60 65 49505 tct gtc tac ttt ttc aac ate gtc aac cct gag ggg ate ate caa ggc Ser Val Tyr Phe Phe Asn lie Val Asn Pro Glu Gly He lie Gin Gly 70 75 80 49553 cag aag cca cag gtg cag gag cac ggg cct tat gtg tac ag Gin Lys Pro Gin Val Gin Glu His Gly Pro Tyr Val Tyr Arg 85 90 49594 gtgaggatgg actgaggccc ccagtgcttg gggcaagggc tatccatggt tggcatgcag ggctgcttgc tgagcacttc cttcgaggtg aaggaaagat aaaaccaact gaaaaggtca agggggttgg ettgeagaag agacttggga etttgettte ggagtggtta taagtgatca cactcaaaag cactgtaggg tggccaaaaa tgctttattt ctaaagattc gggcctttat gctgggactc ggctcattca atggctgcct ctgtcctgca tgaggattgg gtaaatgtta gcaagtgttg aacgcaggtt gtttaaaaaa aggaggccag tgctcaacca cttcttccac aggtcatcac gggccaatct ggctttgcca atgtcccctt atgagcttat gttattaaca ggatttccct tgatccctgg aaaaaaaaaa caatgtgggg tgaaagtttt tgtgtgcaaa cccattttac gcttgctcca ctcagcaggt ctgtaaaatg gtggagccca cattatcatt ggcagtccaa tcaaggaact aaaggcaagt agaaggegga taaagggaga actttgttct agatgaggaa ateeggtget ctctgactat gggatgaccg gcacagtgcc acctgagttt cagctaagaa aagacaggaa gctggttgga ttcacgtcca atcttttgga gattggttgg 49654 49714 49774 49834 49894 49954 50014 50074 50134 50194 50254 50314 561999 70 tggtgcagtt aacagggagg tgttctggga gtcttctgct tggcagaggg taccatcctc 50374 caccttggtg ggggccttag ttcctgcaga agaactcaga gatactgttc tgtgtatccc 50434 ttgaggagga gccaggaccc tgccccaagg ctgcactatt ttttcctgac tgcccctcct 50494 ttgcttctge gtcccctccc tcccctgata aacaactctt tgaatctgcc ccctggaact 50554 cagggaaggt caaagaggct gaatgaagtg tattccctac acaaaaactg gggacacgga 50614 aaaggatttt tacctgggag ggccccacag ggtcctgetg tctttcttta tgaccacagc 50674 tgtcacaact ttttcatttg atcattttct ttgggagggt gaagctctcc agtggctact 50734 gggctgcctg gcattggctg gagcagtttt ccaagagatg ctctgccaca aggctccaga 507 94 agagctgcca ggtgccaacc tgcatcacag actccacagg gacattgggg aaggggctcc 50 854 ctggaataca agacccaatt tctggtgctg cccattctgc cctcaggaaa ctgcaatcct 50914 ggcttagctc ataaagactt ctgcatcgaa getgagcctg gtttgccaaa gttcttactt 50974 atctcttcat ttcataagcc tttatgaagt actgaacatc tcctgtatgc cagacactgt 51034 gccagttcac tgttaaaggc agacaagagc ccctgcctta cagagctgac attcccatcg 51094 ggagagaatg aacaagtgaa cagatgaggg gaaacccagc agcataagcg tggtgccaac 51154 aaagcaggtc aagccgtgga gagtgctgga ggcagggctt ggcgggggct ggtctaaetg 51214 cagggtcagg gaggaccttg aeaaaggggg acacttccct agacccagat ggtgagaagc 51274 gggcactgca gtgtgaggat ctggggggaa gtgagtcctg gacagaaagc agcgggtgcg 51334 aagggcctga ggcgggaagg tagtctagga ggggagaagg cgtgtgtggc tggtgtgtta 51394 ggagcgaggg gagagtgagt gggagagagg agacctgaga gttggacggg gccggatggt 51454 gaggaccctg gagcccacag aaggagctca gttttatcct gtgtgcaaag gagggtttgg 51514 gggagaggat gacgtgatct gatttcccct tttcaaagat tcttctggat gctctgcaga 5157 4 gaatggattg cagagcatga tggaataaag agaataacag caataaaaac tgcagctgat 51634 acatgctttt ctctgtgcat taggcaatcg ttttagttgc ttgacatgca ttagctcact 51694 caattctcac aagtagggac ctaatgacat aagtctgctt attactccca ttttattttt 51754 tttccttggc tgtacctcgt ggtatgcagg atcttagctc cccatcaagg gattgaaccc 51814 atgctccctg cagcggaagc atgcagtctt aaccacggac tgccagggaa atcccattac 51874 tcccatttta aacatgggaa aacaggcttg agtagtaagt gatggagaca ggattcgggc 51934 cccctacctt ctgtcttcag agtgcatatt cctacccacc atcctcgccg cctccctcag 51994 aaaagctagg atgcccaggg ggagggtgat gagggacgga gggatggtgg cagcttgccc 52054 agggggaggg agcatatggg atttggtgac aagaggcttc tgtgcttgtc ctgcagtagg 52114 tgggcttccc aggtggctea gtggtaaaga atccgcctgc cgatgcagga gacccaggag 52174 acccggcttc gatccctggc tcaggaagat cccctggaga aggaaatggc aacctaatcc 52234 agtatttttg cctggaagat cccatggaca gagaagcctg gcaggcccca gtccatgggg 52294 tcctgaagag tcggacacaa ctgagtgact aatactttca ctttaagagg atcctggagg 52354 gctacataca gtccttgggg tcacaaagag gcgactaagc acataccaca gtgggtgggt 52414 ggtggaacag acgtgtagga ggcagtcaag cttggcagac agacagccga ggggcccccg 5247 4 ggctcctggc agctgaccca ccagcttcet cccccacag g gag ttc agg cac aag 52529 Glu Phe Arg His Lys 100 age aac ate acc ttc aac aac aat gac acg gtg tcc ttc ctg gag tac Ser Asn lie Thr Phe Asn Asn Asn Asp Thr Val Ser Phe Leu Glu Tyr 105 110 115 52577 cgc age tac cag ttc cag ccg gat aag tcc cgc ggc cag gag age gac Arg Ser Tyr Gin Phe Gin Pro Asp Lys Ser Arg Gly Gin Glu Ser Asp 120 125 130 52625 tac ate gtc atg ccc aac ate ctg gtc ttg gtgagatacc cctggtcgtc Tyr lie Val Met Pro Asn lie Leu Val Leu 135 140 52675 tctccgcctg gaeteggegg ggcttttccc ttccactgca tctagactct tgtggtgttt aagtagctgg gttcactgac ccccttgcac gcttcctggg ttttcaatct ggggtacgcg ccaaaaggga ggagggagat gcttactgcc teattegtag tgggcggtcc tgcaccttgg gcaaatctag ggttaaaccc gtacttgtgg tgtggacagg tgcctgccat cgctcgcagg cttgagggct tctctggttg ggacttccct ctaatcgtgt gaagctgaat atgtccattc gtgtgccggg acccggcgta caatcggtgg ggagcattgg ggtagtccag gcaacaacaa gggggtgctg tetgeaggta gcccccaaga tatggttgtg ctcctgggtt tcaaagtcat tggttaagac caaatacaaa tgtgcttgtc agagtcacag ccacccccag ctcttggctg 52735 52795 52855 52915 52975 53035 53095 53155 561999 71 tagtttggta ggtgaccaga cggtgattta tcagcgccta atctgcgttc aggaagcggt tctagagaga ctagtgtgtc ggtactatta tatctaaagt gaggctgcca tcagcagctg cctgcgaagt ctgctgcctc aattaggttg gacactgtcg tgaagtgatt aatgtgtcct atttcagcaa atctactgca gggtgtcgtg tgcccctact cacgtgcttg gttttaggct gcaaaagcag ggcctgggtt tgcatgcctc aaagaagccc gtggatatac gtttttcttt aggccatggg cctgcaggaa aggagggtca catctaagag acaggtttga nnnnnnnnnn ttcccctttc gtctgactat ctccccgcaa agggattgaa gaacccaagt gaatcgaacc agctcagata ccccgtgaat gactcacgtc tcctcctgcc tgaggtggcc ggctgatctc gtcttcttca tccaactctc ttgttggcaa ttccaaggag agcctagaaa tgatgggacc cgctctccac gggtggtgtc cttgtgtttc aggatgataa atgtccagtt gtggtctggt gagtggaagg cgcaggcttc tgaccacaca gtgggtattt caggaaagca gggagccctc actgtctcag aggcttcaca ttttctgttt cactcaacta ctctgtggca tgtgccccgg ctgttgggtg cctcagagtc gcaaagactg ttaggaacgg ttcgatgtaa cttggtagaa cgttttcaga gacgagcagt aagcagtctc tactgtttct tacatgcctt ttgagggcta gggcttccct cgacccctgg aactaagact aagacaaaas tgaaatgtga caaccatttg ttggctttgg tactcaagaa caggttagag acttccctgg tctctggccg nnnnnnntct tacaggtaga ttgtgaccgc gaatactgga cccaggtatt atactggagt aacgtctcct ctggatgttc tgcagcacgc catcgagtca cccaatccct aaagtactgg cttcagaatg acaccacagt acatccatac agtaatgtct taagcgtctt aataaagtct ggatgccatg tttcactttc atctgcatat ttccagtcca tatacagcct ctaactgttg attcccatct aggcagaaga cagagccacc caggtaggtc attgggggct ggtggtacag ctaacatcag gtctgctgtg catactacct gtagaagagg gcaggtagca ctcagcctcc ggctggcccc gacaggatga agatgtgcgt tgtgataaat aactggtatg attgcatgtg taacacatgt gcaaacatct ggattagtat ttagttatgt gaaggagaaa gaccagctgg gatgttctct ggtggtccag tcagggatgg tgaagcagcc caaatgagtg gtttcatata aaaacataca cccgtggacc cccagagcag agggactttt tggtccagta gggaaccnnn atggaatcag gattagatac atggactgta gtgggttgcc ctgcattgca gactagcata gctttgcagg agttcagttc caggcctccc gtgatgccat cccagcatca agttgcagct gactggttag tcaaaagcat atgaccacag ctgcttttca ttaatttcat gacactgctt atcttcgttt atcaagaggc ctgaggttat gcgtttctca tgacatactt cttcctgacc ctttcagaat gaatccacag tcccagggga tcccctacca tgtcacggag tccaggcaca gatctcagac tgagcatctt gtttacttgt aaaactgaag ggttagggtt cattcaatcc aggctgggct aagctcagat gtttaggcag gcccagggcg agctgttgga cccctgactt gggcaaagat actgatcatc acatccagca ctaactaagg tacttgcatg gctttttcta gttgcagctg tggctaaggc cgcctggtta aaataaataa ggcgtggcta attttcatgt gaacatctta acggcttgct tgggttgtct actgtatatc gttaagaatc nnnnnnnnnn acctttacgt tggatgtgtt gcccaccagg atgccctect ggtggattct ttccttctcc cagtttcttt agtcgctcag tgtccatcac ccagccatct gagtcttttc tcagcatcat atctccttgc caattcttcg gaaaaactat atatgctatc ggctgcagtc ccactctttc tctgaacgtt ttttgagttc tgatatttct tgatgtactc cttttcctat tgcatacaga tttccagttt agggaaaggg gtcacgcttc ggaatgctca acgccctctg gtgaacccct gccagcaagg gctgacacta cagcaatccc aacagagagg ttgaaactag tctgtccacc caggatcaga aaatacagcc tgaagtcact taggagcgtt aggtaatttt cagaagtttc gtttgttctc atggggagtt aaaggaggca aaaatcgaac catgcacaca taaagggcca ttcaaatctg tccgagctcc tatgctgtaa ataaatattt tatgccaaga ttttcatacg gctcctggac gacttctggc tgggggagga cttttacgat caccttgcag nnnnnnnnnn ttaagaaaat tgtgcttatt ctcctctgtc gcaagggatc ttaccatctg aggggaactt accagctgag tcgtgtccga caactcccgg catcctctgt caatgagtca tccttccaaa agtccaaggg gcgctcagcc agccttgaat taggttggtc accatctgca cccatctatt gagctttaag ctcttcactt cccgacaatc tgcatataag ttggaacagt tttctcaaga attgtgatcc cacgtgggat attccttgag ctacagagac cctggcacgg cttatcagtt gccaggcctt cgtgttagaa acgatcatta ttaggtgact tttggtctat cagttgaggg tgtgaatgca tgaagccttg ttcccccacc tgtgggaaca caacttaaat cataggtagg aaggatgttt atgttgtgat gggctgcaga acaagcatag ctcacttaca gataacagat ccattataat caatataggc tgaaggatcc ttaaaatgca gacctatttc gtcttcttct tgtaaaaagc gttgatgttt gattaggaag ttttaaaaac tgtagggaat nnnnnnnnnn gctggttccc gttcagtcgt cttggggatt ttcccaaccc agccaccagg accaacccag ctaccaggga ctcttcgcga agttcactca cgtcctcttc attctttgca gaaatctcag actttcaaga ttcttcacag agatggacct ataactttcc gtgattttgg tcccatgaag ccaacttttt tctgccataa ttgattccag ttaaataagc ctgttgttcc ggcaggtcag acacagtcat 53215 53275 53335 53395 53455 53515 53575 53635 53695 53755 53815 53875 53935 53995 54055 54115 54175 54235 54295 54355 54415 54475 54535 54595 54655 54715 54775 54835 54895 54955 55015 55075 55135 55195 55255 55315 55375 55435 55495 55555 55615 5567 5 55735 55795 55855 55915 55975 56035 56095 56155 56215 56275 56335 56395 56455 56515 56575 56635 56695 56755 561999 72 tggatgaggc acctttaaaa agtaaaccaa gtgccaagat ggtatctcca gaggaaaaca 56815 aaaattcgaa aataaattca cccatattct aagcacccag atactaggta gaactggtgc 56875 ttggggctct tggagtttgg aagggagatg ctaaggaagt agagtacagt ctgcattaag 56935 attctttgcc tataaggtga cagtgaaagg tactctaaat tggcgtaagt aaaaagggca 56995 tttatgggct catgttactg aaagggcagg agttggcctg gtttcaggtg tggcttgatc 57055 cagagtgcaa atgaggttgc ccagatttgt gtcggttcca tgttctggta gatccactcc 57115 tcagggtggc tgcagtctta cacacaccac gttcaaaggc agccagagcc accttccctc 57175 tctaccccat aaccccagct ggtttgctcc ttgtggcttg ttggttctga tggggctgca 57 235 tacatattcc cagaccaatc actgaagcca gagagaggaa ggtttctgat taaccagggc 57295 taaggcacac gactgcctct ggggctcggg gtggagttaa gtcggcctga acccgagagc 57355 acaggtggtc ctgttactga aggatcaggg gatggttccg gatgccccag atgcccactg 57 415 cagggatcct tctaggcctg accacgtccc gggatggctg tgccttgaag cttggcttga 57475 ggtccaggga gggcagacaa gctgtgtcta gcctctaacc tgtggtgtcc cctggctctg 57535 cag agt gcg tea atg atg atg gag aac agg ccc ggg ctc ttg aag ctg 57583 Ser Ala Ser Met Met Met Glu Asn Arg Pro Gly Leu Leu Lys Leu 145 150 155 atg atg acc ttg gcg ttc age acg ctt ggc cag cgt gcc ttc atg aac Met Met Thr Leu Ala Phe Ser Thr Leu Gly Gin Arg Ala Phe Met Asn 160 165 170 57631 cgc act gtt ggc gag ate atg tgg ggc tat gat gac ccc tta ate cac Arg Thr Val Gly Glu lie Met Trp Gly Tyr Asp Asp Pro Leu lie His 175 180 185 57679 ctc ate aat cag tac ttc cca aac teg ctc ccc ttc aag ggc aag ttc Leu He Asn Gin Tyr Phe Pro Asn Ser Leu Pro Phe Lys Gly Lys Phe 190 195 200 205 57727 ggc tta ttt get gag gtaagtgtgg cccagtgaga agttcagggc cgctgtcttg Gly Leu Phe Ala Glu 210 57782 tcccccactg ccaggagctg agtttcagtt attcaggcac gaggttggtg attgetttge tcagtgattg ctgaccttgg tttccttttg cagttatagg gtgtgggtaa actggtaaac taggtatagt cttcagggat tcaagattac ggtagcagca aaagcaacat ttccgttagc taacttacag aggggagaat atgatagtat aggctagcat taggtctttt tggctgaagt ggaagctaga agctatttgg tacggcatcc acacagcctg tgggttgtca aaattctatt agcttgatcc ctggaagaaa tctgagttgc ggatcctgag ctccctcaat gggctctgct catgtgatcg gttgacaggt aaatgaaagt ggaaattcca ctagaatttt tgcatggtcc ggaaggaaga gccctggctg cagaactaga ctagtgtgtt ggactctggg tattttgggt ttggagatgt aacactccca ttgggctctt gtaggctttg ccggtaaaca tcagaagtaa gaaaccagtt gtgacagaaa tattgattta aggctcaaat ggggaatggc ctgctttatc tcatgatctg cacagattgt gatctggtgt ctgctcagta ttaaggtaag tattagagat aagcaatagc actactctgc aagaggctgc agtggtaggg tttccctctt cctgtggcta gatgctctga gagtaactag gagcatttat tactatttct gtgtctcttt aaagctctga aacttaggcc acccctactg aagggtgctt accagcttgc acctaactga ttggttcata gatgtgactg tgctggggag ttaatcgtga atgtagagtt aaacagccct tgggggtagt catttttgag tgacatgatt tgagtcacat ccagcagtgt tctccccacc tggggcacca gattcagggg tgggaeggag cacttagctg gtaaggtttg aagtctctga taataataat taggcactat gtagtggtgt atggacaaat ctgttggctt agttcctcac tctaaatttt ttttggagct aactggctta taactgaaaa gggctgagct gcaggtcaaa tccctcataa ggatccctga ctgagttgct aggagggaga caggatgaca cttgtcaaag gcttataaac tggtgtggtg ttagagtgtg gctatcacac cagtcggggg aaaaccaatt CAagggaagc ggttttgata tacacttggt aacagcaata cctgaacagt ctgttactgt gacagggcac acccagtcag gggtgctgta acccagcggc ggcagaggcc agccaaagcc ctgtagggag tagatggttg aatgccaact cctagtggtg cctctgatcc ccagcctgag aatcatgatt ggttctgggt caaactgtaa tatttttgac gcttcccact gtttccatac tcactttcca agttgaggac tccacttgtt tggaaaatgt tgaaggagaa tataattatt ataaaaataa ttttacacat ctctttatac actgatatca taatcaatgt ccttacatta cactgatttt 57842 57902 57962 58022 58082 58142 58202 58262 58322 58382 58442 58502 58562 58622 58682 58742 58802 58862 58922 58982 59042 59102 59162 59222 59282 59342 59402 561999 73 tttgtgtgtg ctttgcacag ttttgctttg aattagaaat ctttttctaa gaaattccct 59462 cagggtgcca aagcctggac tttctgtctg gaaagaactt gataaacatc aagttctctt 59522 tgcctgtatg tttcaggata ggccgggtta agcagccccc agatctccat ggcttagaat 59582 agcaaaggtt aatttcttac ttgtatgcca ggtctagtgt gggtgaccag ggctcctctg 59642 ctctttgtgg tcactcaggg acccaggctg agggaggctt ctgctggata catgcttctc 59702 tggtctcgat agcagtgata gaaagcacag tgacatgtgc atgatcagct cttaaagctc 59762 ccacccggaa gtgacattat cctttctctc ctttttcact gatcgaagag gtcacgtggc 59822 catgctttct tgtaatttgg tgaatgggat ggggtgaccc taccgttcac ctgagaggag 59882 aaccagactt tcctggagtt aggaagggaa aatgggacgt tttctaggac ggatgtggtg 59942 ttcagaggag tgtccctcct agaaagccct caagtcctcc tcctctcgac tcccccagtg 60002 ctggacttgc agcggtgaac aagacaatga ggcccctgtt ctctgggtga agggggagca 60062 gcctgtgggg ggcctgagta ttgactcccc atgtccttta cctccatcct ggggcttaaa 60122 gatgtcattc tctgctgttg tctgcag ctc aac aac tcc gat tct ggc ctc ttc 60176 Leu Asn Asn Ser Asp Ser Gly Leu Phe 215 acc gtg ttc aca ggg gtc aaa aac ttc age agg ate cat ctc gtg gac Thr Val Phe Thr Gly Val Lys Asn Phe Ser Arg lie His Leu Val Asp 220 225 230 235 60224 aag tgg aat gga gtc age aag gtgagggcaa gaggtgacag ccatcctgtg Lys Trp Asn Gly Val Ser Lys 240 60275 cagggaccag taaaacagga aggatctcct aagaatctga tgaaactaac gggattgeat aactagagaa aggagcctgg cactttcact agaatcccag ngaccccatt ctgaagatac aacacaggaa tgtatgtctt tcagtattgg tttcttctag acctgagtgg ttccaaacgc tgagggctgg ccagtaataa taaaaatgta gggggcccgg caaaatatgt ettttgegae agaatacctg acctgtgtct tatgatgtac aaaaaccttc gtgtcactct ttaaatattt acttcctgtc tgagtgacag gggacgcacg acagcctgat tggtgacttc tttatgagtt atgggggtgg gtttgggatt gtgtagcaca aaaagaatag acaacattat ctgtagggga ggcaatggca taggctgcag ttcatgcatt annnnnnnnn gtttctttcc agccatcctt gacattcaca cacacgccca gtatattctt gagcagaatg cctttcagca tcatcagggt ggacagatta ccattctgaa cagggacttc gttcagtctc gtgtgtgtat cccatggatt agtgggttgt cctgcattgg atatatacac tactgggaca ccagaagcaa tacttatgga tgagaggega gtccgcttta gtcagggaat ctccttgcac acatggtgtg ettgettgae caagggctgg gacgtgtaca gggagtgctg atacatgtgt aaatcagcta gatgctcttg ccccactcca tccatggggt ggagaaggaa nnnnnnnnnn tttggtgctt tgtactaaaa ttaaaaaata gccccattcc cctgtgtatt aaatcatact tttctcatga gaeggttgea tcccccatca tggacactgt tcaggtggtc tggtcgggga atgtatgtgg gtacccacca caattccttc caggtggatt acacacacat gatgggtaaa ctacggtttt actaccttct tgatgtctgt cagatgaagt ggggagctgc caggacgtct tgaagttgga taagaggctg gatccaccgc cactgctata ctcaatattc atgtaaagtg tgcttcagtt agtcccagac gtactcttgc cgctaagagt atggcaaccc nnnnnnnnnn cacttacaag gattgagtaa aaataataga cacccgaact tttatccatt gtgtgcattg aaatgattat gagagggctg gggcccggac gggaattctc cagtggttag attaagatcc ttgttgtgta ggctcctctg tccagaggat ccttgccact acacacatac aagtgagaag tgtaatctcc cacttgactg ctctcccgct tactgagtcc catcgatagt atccaggagg gctccaagtc tgtttaaatg ccagagcacc tttaaaacag cttaataacc gaatcacttt aaagaaacaa cttatctgtg ctggaaaatc tgggtacaac actccagtgt nnnnnnnnnn aatgcttcaa aaaaaaaaaa gacagatata acagaggaaa tgaggtgcga ttttgcaact gtggtggtgg taatagatta tgacaggatt tttggcttgc gactctgagc cacatgcagc gtctctaagt tccattggat cttcccgacc gagccaccag gcacatatga tttttttttt tggtagegae ttttgatgac cactgtgctc ccagcaaagt ctctctggct actggcttct acgtgggcag cttccccaca ttcttcctct acaaccaacg taaatgggaa gctgtacgcc aacagaggag tttaaggctc ccatggatgg tgagegaett tcttgcctgg nnnnnnnnnn aaatattaca ttgcactaga aagtgcactt cccacagtta tttttttttt tgetttttte tgcccacacc tcctctgggc accatcgcaa tttctcaaga ttccactaca atggtgtggc catgtcaatt ttcccaggca cagggatcaa ggaggcccaa acatatgctt ttctgtccga cattatgatt gtgtctgccc cttggaggtg tgtacgttca gtctgtctac ctcaattggg tgtccaagtc g gtc aac 60335 60395 60455 60515 60575 60635 60695 60755 60815 60875 60935 60995 61055 61115 61175 61235 61295 61355 61415 61475 61535 61595 61655 61715 61775 61835 61895 61955 62015 62075 62135 62195 62255 62315 62375 62432 62480 62528 62582 62642 62702 62762 62822 62882 62942 63002 63062 63122 63182 63242 63302 63362 63422 63482 63542 63602 63662 63722 63782 63842 63902 63962 64022 64082 64142 64202 64262 64322 64382 64442 64502 64562 64 622 64682 64742 64802 64862 64922 64982 65042 65102 65162 65214 561999 74 Val Asn tat tgg cat tcc gat cag tgc aac atg att aac gga act tcc ggg cag Tyr Trp His Ser Asp Gin Cys Asn Met lie Asn Gly Thr Ser Gly Gin 245 250 255 260 atg tgg gca cca ttt atg acc cct gaa tcc teg ctg gaa ttt tac age Met Trp Ala Pro Phe Met Thr Pro Glu Ser Ser Leu Glu Phe Tyr Ser 265 270 275 cct gag gcc tgc eg gtaatcgccc tgatgtgtga cagggcctcc ttctgctttg Pro Glu Ala Cys Arg 280 tggatgetea ctgcatgtcc cgaactccaa ttggtggtgg gatgggctcc ctcttgcagc ttccagtggc tgtttccctg ttcactgatg tetgegagag gttgtgtcct gtgtttctgt tcccagaaag tgattctttt caggcaacag nnnnnnnnnn gagctgaacc actggcccct ctggtggtgc tgatggacag ctgggccagc ccctccactt cccggcagag tcctcggctc tgaattcaag gcactggggt ggcaggggac tctgtgtgtg tgcagttttc gtcaccccgt tactggagtg nnnnnnnnnn aagtggcttg ggcgcccgct ttttgeaagg aaaatgggca tgccctgttt tccagagtac ggatgagaag ctctgttgag agactcacct ccctgcagag tcaggcccat ttcacttcct tetgeatttg ggactgtagc ggttgccatt nnnnnnnnnn tggcgtgtgc tgaattctct cccagggagg agatggcaag ccagttgggt agcctcggag agacagcggc gccactctgg gccaccctag acaggatggc ggacctgcag gaaattagaa ctgttgttta ctgccaggct tccttctcca nnnnnnnnnn ttggaaagcg ttggctgctc ctgctggcgg gggccgtgga gctgagaggc gtcagcttgg tcctagagcc aaaaatcctt cagatcttga cgggtccttg gggagggtca atttcaactt aaatgtcgct cctctgtcca ggggannnnn nnnnnnnnnn cctggctctt catgaagagc ttgttggggc ataaggcaac ccggatgcct ggctaaacaa gttctctggg tgcttgggat cacagccctg gtccctgggg gagagtggag agagaagtgt aagtcgtgtt tgggattctc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tcattttaat taaacaaatg acttccactg gtgtggcaaa tgtttaccca cacatcccaa ccccgtggat gcctttgagc tgctccctgc ggaataagag ggagggagca gggttattgt cgctaggttc tcagtgatgc tttcccagca tggagcttca attgactgat ggggctgaat ggaagtagtt tgtggagaat atttgccagg tgtggggagt ctacatcatc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn gatgttttat tacaatacta cagggggcat aaaaaaaaaa attgtttgga cttggactgg aggacaggtg aataaggcca ggaggtgaca ggaggaacat tgtgggaaga teagttgeta ctctgtcctc tgtgaaacca tcagggtctt gcttcagcat ttgatctcct cctatagaag gcagtcttat ggattggtgt gagagaegga gagtttactc tgtccctgca nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnggctgcat taaggaaaaa tcaggtaaag tggacccagg agatccaaaa tggtatcatg tcacagtggg aagacggact tgcctgggcg tccagtggtt gagactccac gggttctgtc cctggccagg gaagttccac aaactgtgca aaaaagagag agagaaaaat attgaagagc tgttttcaat ggtatcaagc cataggaatc tggagtgtat ttgacatagc ccccatttca agggctccaa ggccatgtat gctgagtgga ggctggttct catccacagg ccatgttcaa gtttagaagt ggtgaacgga aagagggtga atgggggtgg gtgatggtgc gcagaaacgg gacctgagtg aagaactgaa gtctgatgtg tccaggcact gggagcagca ggtacaaagg ccctggggca atggccagga ggcccctggg gtgcagtaca tggtgttgcc aagttgtgtc cgactctttg cgaccccacg gaetgeggea cactatctcc cagagtttgt tcacattcat gtccattgaa tctcatcctc tgcggccccc ttctcctttt gctttcaatc ttccagtgag tcagctgttt gcatcaggtg gctgaagtat cagtccttcc agtgaatatt cagggtggat ttcctttagg tgcagtccaa aggactctca agagtcttct ccagttagca cctgtggcca tggtgatgac gttggatttt attctagact gcggggttgt tgtgatatat tttgtccttg ttttaaagct aggtggtgga ggcagggaga cacataagga ggttgttgta gcaaactggg aegaggeggg ggaacggggg tggccatgcc aggccaccca gttccggtca ccgcctgctg ggatgtcatc g g tcc atg aag ctc gtc tac aag gag cag ggg Ser Met Lys Leu Val Tyr Lys Glu Gin Gly 561999 75 285 290 gtg ttt gga ggc ate ccc acc ttt cgc ttc gtg get ccc age ace tta Val Phe Gly Gly lie Pro Thr Phe Arg Phe Val Ala Pro Ser Thr Leu 295 300 305 65262 ttt gcc aac ggg tcc gtc tac ccg ccc aac gag ggc ttc tgc ccc tgc Phe Ala Asn Gly Ser Val Tyr Pro Pro Asn Glu Gly Phe Cys Pro Cys 310 315 320 65310 egg gag tcc gga ate caa aac gtc age acc tgc agg ttc a gtacgtgtcg 65360 Arg Glu Ser Gly lie Gin Asn Val Ser Thr Cys Arg Phe 325 330 335 tccctgtggg ctggcctctg actcaaaaaa ttggtggcct acacaggcct gaaggggtgt agttgtgtcg gctgtctccc cttatcctct tccagtgagt tccaatgaat gaagggactc tcagccgcct aaaaaccata taccttgtct getgeagtea agcttccaga gcaacatcag tgcttccttt aggccacact tttgtgaccc catcattctg tttcccctca teagegtegg ctcaggttcg aaaaaaccac ttgggtgtgg tgtgccagtc caatctccct ctgagtcctt ttgcattgtg tgtggctatt tccaaggggt tggccagaaa taaaaaagca gagtcgaatg ggaagttcta tggcaaccca gggggcccac tactttccce agtttggtcc tgggtctttc tetgatttge ccctttatta aeggtaaaca tggggactca agggegggag tacctttgat aaaaaatgtg ggaatagcac ccccatgcta ctctgtttcc gactctttgc ggagttttct gccacccact cagctcttca attcaggact tcaaaagtct ttatggtcca gctttgacta aggtttgtca ccatccgcag gacccctaga agctctctga ttccagtttt cctattttag ttgtaatcca cctccccggc tctggggagg acttccctgg atccttgatc aaaagataat ccgctggcgc ctcgaggcac atcctgaccc cctgtcttcc gctaccctct taaattgaaa gtgggttcga aaaaaaaaaa gtgccttatt actgtcctat ttggacatgg accgttttct tgttgtcggg ttaactattc tgcctcaggg cctcacctcc cactctaaag tcatcctggt ctgtaggcag cgcagtgtgt gtggggcccg aattccatta ccgtgtgtat aggtttacga agagccaggt ttgccttttt aacctcatgg ctaattcatg tctcctcctg catcaggtgg gatttccttc tctccagcac actctcatgt gatggacctt tagctttgct tgacttggag attcctcgct tcccactcct aagggccctg gctgattggc caggtcctag catgatgggt aaaggaagct ctgtccagtg aggaaactaa taattaaaaa tgccgcattg cccgcctcat tcatccaggt tgctgcctca gtgccaccca teggtgaett ttcctggtcg agaaatcaat gcattggcca gggatgaaca ttccagccag ttaaatgtcc atgtggcctc tttagcccca cttttgtgtc tcctggtcta ttgctctccc gctcatcacc gtctgtgtcc aaacgtaggg tgccgggggg gtccagagat ctgttgctgt tcttaaggac gtcagcaggc cagcttctag actgcagcac tccattgagt ccctcaatct ccaaagtatt attattgact cacacttcca gtggatggtc tgttggcaaa tccaaggggc ccccagaaaa cagggttccc gtggtcacat gagactctat agccctcacc ggcttaggaa agcttaaaat ggcaacggcc gttaaggctc gatccagcat aaacttgttc tcctctagca gtgcttactg cactggcaag tggtccaggg tttctcagaa ccctggtggc gggaactaag aaattgaaat tatttcagat gtacagaaag ccgcccaggg aacccatgcg tgctggtctc ctgcctttgc agctattccc ttccaatggc caccccacct ccctgaaatc tcagcttcta atgtgtgagt cttgtctgtc agtgaggggc cgtaataaat tcctgtggtt tgcattcctt tttttgttgt tccagtctcc cggtgatggt ttcccagcat ggagcttcag aatttgatct aagcatcaat catccgtaca gtgatgtctc aagcatctct gaaaatctgt ttcctccatc ttctttttct ctggcctacc cctcctgtag gcagacatct gaggcatcgt tgagttccgt cgcacttccg gcctgtcgca agtggcgtgc tcgtcagtgt tggagacttg cccggccttc cccttcagtt ttegcagtgt ccggtggtta atcctgcata tggtttgatt ggtccccacc agaatatttt ggcctttccg tgtagagega tctaatgtga cctctttcta caggctgctg acctcctcca cccaaatact actgaaagtg gaattgtgct gtatcttact tetttaatge caggaagata tcccacaaac tagaagtccg tctggaggta taagtcactc tctgtgcttt atctaaccat cagggtattt catcagtcct ccttgcattc tcttcagcac tgactactgg tgctttttaa taattgcaag cactgcttcc ttcaaaacca gatacatttg tggacaatct cctaattccc ttgggaaggg agggaggacc gaaatcttgt ctgcaggggg tggcatggcc agttacgggc tttctggttg gatgtcagcg aaattaattc ggccttcctc gttcccctta aggcttccac tgacatgaca gaaatcaaat ttcatgtgct cattatcata agactctgac agtccttaca tttcttcccg agatatatct tagatctcta gcaggcctgc ctgtctttca tctccactgg eggtatgtag taggaaattg 65420 65480 65540 65600 65660 65720 65780 65840 65900 65960 66020 66080 66140 66200 66260 66320 66380 66440 66500 66560 66620 66680 66740 66800 66860 66920 66980 67040 67100 67160 67220 67280 67340 67400 67460 67520 67580 67640 67700 67760 67820 67880 67940 68000 68060 68120 561999 76 aggctcaagg atctttctag tggggatttg acggtttccc tgcccatttc cttcactggt aggctgtgtg cgaaaagggc tccaaagctg gggaggcttt ctcctctgta tgactggctg caggtgctca agtaggggag caaacaacat gcacgtgtgc aattttttct tttgaaagtc acttccagca gtatttcttg cccattgttg tttccttacc tttagtagcg gtggtatttt ttcttggact tgctcactgg atgtgtcata tgtttggaaa agctgaagca ttgcaggcgt gcttccctta gtagagatgg gcccaagacc gggggtcttc gaggggaggg tccagtattc ggatcacaga ttctgttttc agggcttgag agggtgccag atttgttcac ttatgcctca gcttcaaaga agagatactg ctctgggaaa tggctttgct caggaatgtg tgtgagtttt accattttaa gccgcggtct actccccatc ttgcccattc actgtccgtc ctcggtcggt gggatccgag gagagaagga gtgtgcacac tggcttggca tccatcattc gcattcacct ctggggtgtg gtcttgtctt ggaggggttg caagttcagg tggcatggcc gtctatggga ctacacagtt acttcttggt ccttttgcca tggctccagg gtccagcaaa tgagccaatc gcccctggtt gggcttaatt atccatccac caaaagaact tttacattga cacaaaggga ccacggcttt ttctctactt tgagagctct tcttgtactg ctaggaatgt atctgaagat cagaaaactg agccccttaa aagctataaa taaaatagaa agagcaggtg gatggagctt ccaaggggga gagaatggga ccaaaggttc tggggtggct ttcggtcctt ttgcctggaa gtcagacaca cattcttttt tgcctttcta actcttcttt attgtattta gatagcaagc agcacattat ggaagaataa aatgaagtca aagaaaagca ggtcacttgg gttctttctt agtttacgat agttccagag tttgctcttc ttgacctttg aggcttgtct tgacggagag tcagggtctg acgaagcagg ccgaacaact atccagaggc cttccaggca ctgccgtccc actgagctca ttggggcctg tttgcaggag cagaatgagg gtttctggcc atttctgctt gccatagcaa ttatctttgt tgggccagct gtccaggctc agtcccagag agtggccaag ccagtgagtg gaagcctggg tgccaagggc tcagaagtgc aataatatat tggagaaaga gcctgccttt ctggcttctt tttcattgct ttcatgggca tctcatacat aagaaatacc tctctcctcc ttgtaatcca ttaattacca aaaacatgta atggcaaatt gtttgaggtt ggcgttcctc agcaaggttt aaggaggcag caactggtaa ggctcaggaa aatctcatgg attgagtgtg ctcctcctga acactcatgt tcatgcaggt ttcctgtatt atctttaaaa gtaacagtgc attaatgttg gacagcagtg gaaagaacct ctggcagttg ttttttcgca tcggtgacgt cgttctcatc ccggcccctg ccggtttctg gttgtcctga gttcagcttc ttctcacaca tgtgtcttct ctgatcattg ccttcctctc ctgtctgctc ttcacctgga tagggctggt tttgattaga ggtttggagg tacttcatgt ctcctgccct gtaggtgagc gtggcagatg attaggcatg cgattttcct acctcctccc tttgctctga ggtgggctat gagccagcac ttctaggaga taggcttgag aagggaatta acaagaaaga gcaggagtga ggtctttatg tcacccattt gtatagtatt tccaataaga gtttttgggt cactcatttc aacctccgtg tggtgatcct cttattacct catgtgtaca ggtctagccc catcatcttc gtgtgtttgc ttcaaattgt ctttaaataa agaacccacc gatctcctgg atagaggagc tgcaggcgcg ctatctccag gtcctttttg aacagtctct tattttctaa tatagctgtt agggggtatc agaaacttga aaaccccagc agacatttga atgggctgct tcatggtgaa ttagtacatt aaccccagag tcactgccgg ggggaggctt gcacatgagg aggtatctgg ctggccttca cccctcccgt tattgcctgc acccaagttc catactcact atgccatccc aggacaagaa gccacgttgt agggactgtc ttgaagctgg tttcctctgt tgacagtgca ctataccagc gctccagagc ctggactggt agcagaaaag ttgggctggc gcagattggg ccacatggca tggtgcatac aattacactg cgtatgcatt tacccaactg tcctctcagc taaatggagt tcgaattggt tcagtgggac ttctgctcat gcacttgtgt cctttctggg ctgtgtaagg gggcaattag gggcgttgtc tactattaaa atctcagtct tagggcctgg tccgttgatt gaaaccttgt aattgactta tgccaatgca agaaggaaat ctggtgggct cagatagggt gagaaagtgt acagagaaaa ggatagaatc ttccatcagg gaaataaagc cggatatggc agacaatgga ttttatttta ggttgccagg ggttttaggg gttcctgtaa caaaatgttg gagccccagt ctgtgctctg attcttttcc atgccctggt gtgtgaattc tcccacagac ggacgttagg cgaagacagt aagtctttct gtgcgtttcc ctgaactcct gccattcctc cagctggaga agcaggttgg gacctggaag ttgcttcttt gagacctggt ctgattaggc tcagctgtgg ttcattgcca ggttctgtgg ttatatcaca caggctgtag agaggctgaa ggggcaggca cctcatctta atgtaaatac atatacgcac cgctggcctc cataaagcat gaagtccgtc cgcacttcca tgaccattac gtgtatttct ccttgaggag tcctgaaact gtctggttag tctctcaatc aaaccaagca cccccgagcc ctcagtgact tcctctcatg ccttggtggt catggtgcga ggagatgcaa ggccacctgc acagtccacg gagatacggc tggtgctaag agcaagtctg tacagcattc tgtttaccag agtagcagct aacacatcag tgggaaagga aaccggtgct cacacacaaa gacgagacct tatccagtta tgcagcgtcc gccccttagc tctctgtgat tccttgactc gtttgcctct ctcactcgga cagctgctgg gagacctgag ccaggctgct atcctcagtt tcacctcctt ctgccggcat 68180 68240 68300 68360 6B420 68480 68540 68600 68660 68720 68780 68840 68900 68960 69020 69080 69140 69200 69260 69320 69380 69440 69500 69560 69620 69680 69740 69800 69860 69920 69980 70040 70100 70160 70220 70280 70340 70400 70460 70520 70580 70640 70700 70760 70820 70880 70940 71000 71060 71120 71180 71240 71300 71360 71420 71480 71540 71600 71660 71720 561999 77 ctttgagcca gagcttgtcc atcacacacc tgggtgcact gggcatgacg ctctgcccct gcatcttctc cttgctgggt gaatgagttc gcgggcagtg tgagcaggtc ttgctttcca cttgctttga gggttccagt tgtctagtta gctgtgtttt tctggatgtg g at gcg Asn Ala tttctcctcc tcctggcgcc ctgctgtttg cgtcgcctgt caaacgtgct ccc ttg ttt Pro Leu Phe 340 tttaccattc tcttgggtgc catcttgtca gcctcccttc gcctttgtct ctc tcc cat Leu Ser His ctatggcacc ttggtaaata tcatcacgag acccagctca gacaccccct cct cac Pro His 345 71780 71840 71900 71960 72020 72070 ttc tac aac get gac cca gtc ctg gca gag gca gtg tct ggc ctc cat Phe Tyr Asn Ala Asp Pro Val Leu Ala Glu Ala Val Ser Gly Leu His 350 355 360 72118 cct aac cca aaa gaa cat tcc ttg ttc ctg gac ate cac ccg Pro Asn Pro Lys Glu His Ser Leu Phe Leu Asp lie His Pro 365 370 375 72160 gtgagcccct tcaactttct ggttcctttg tagtgeaagg cagtaaatta ccaacatacc tgggttcggg tcagacctgc gggcctccat aaattctttg tggaagctct tcttagccac tttcactaaa atgccttcgc ttatatggct actgcacaca ttcatccaca ttcccatttt tggggttctt gcggttagca gtggttgaga aaaatttttt tgtcttttaa accgcgtcgg taeggagtet tagattagtt tttcccctgg gtagcttacg atgacctgta tgttctatcc cagctgatct ctcgaggatg gacaagtgta catggactgt gtgggttccc ctgcattggc agtgattcac ctatacaaac gaagtgggta attaaattca aaccagtttt gcactcccaa ccatgcggta gcctgtcagc cctctccctg gcattgatag actttattat attaggagtt ataaagatct ggaatttgtc acacgtgacc tcactgcaaa ggtttctgtt tctccttgat tgactcaacc aatccagact tgccgagggt tggccaaatt cttcacattc tgcaccaact acagatgggt ggctccatca cagaagtact gtaagcaagg tttatttcaa aaaataatat atcttagttc taaccactga ttaggcctac caccaagttt ctaggcttca tccaccattc atttatcccc tttgtatctc taacctttta taataagtgc agtctgccag gtttccttct aggcagattc aatttttaaa aggtctttta ttaatgaatt gtctttattg taaaaggctt tgeaagegae tggccgaaag ctgtgggtgg ccaggtacca cttctaggct ctctttataa atcaacctgt ttagataccg ctgtgtgatg ctcccttggg tttgcccttt tctggagtgg gctttttcat cacagcccct gggaattccc gtgtcttcaa aataaaataa atatttctag catttaattt aaactgcagc gggaaagagc tatatctcag gataaggctg aatgtttttt actttgtttt ccttgaccag acctccaggg agataaactg gttacaatgg ctctgtgttg agtctcctaa cgactacctt tatagttctg aaaaaactga ttgtgctcag gctcctctgt ccaggggatc tttaccattg gtttatacta atgcccacta aagttgatca ttagacatgt ttattaggga acaggcttga aaaaaacaaa gggtgggggg ggctgttcca atcaagtcat gggaactgtc aaattagcca acaagaacct ttttgtctac catgtcttta tggacttggt acattcgtgg gctgttttcc agcactccac tggttaggga tccctgtttg aataaatctg aatgctcatt tcataatagt ccaaagaggt tgttctgtcc taaacatggg aattcaaaca ctttaaacct ttgatgttgt ggatcaaacc aagtccccac agcagaaaat atgaatgaat ttcaggttta gaatggtttc ctacattcac ccttttccag agtataatta ttgcttcagt ccatgcgatt ttcctgaccc agtctcctgg cacttacagt ccttctgtcc taattaatat taaggctttt cttccctggt tccctggtca ggcttttatt aatttctgtt gggtggggat caaagtttgc tctataaaaa gaggcagccc ggcacgtctt aaacagggac ccctgttgct ggctttatcg tctccctcca ttggtgtact cttctagctc attccagtgg gggaatgaag ggttagtgaa atgctccaga eggggaggtg taagtgctcc tgttaattgc tttgatttac taggtcttct gagtatcatt tttgtttttt cttgccccct tttgtttttg acagagttcc gttgttatta tgggttttga actgccttca tcccatcaaa aatgtcatat atagaccgta cgtgtctgaa ttccaggcaa ggggatccaa gaagccccag tacttaattt tgtattaata gattctatta ccatattgtc ggtcctttgg gggaactaag agaaatctaa tggagcgcac gggaagcctg cccgactcca gatcataata tctggctgtg gcagataaac caggagcata tcttctctta ttagttaaaa taggcctttt actcagattt tctcttcttt ttaggactcc atcccgaaaa gtcagccatg cataatccta ggcattgcca agctagtaaa ttgaattcta tgagatccta tttttaatta tgcatatgtc gtttgaccat gcagtggaag ttttgttttt tagacctcgt actaacqtca caaatgcatg gaatcccgtg cccttggcag agttggaacc ttattcatat tgtgcggccc gaacactgga cctgggtctt tgtataatac aaagtattga ttcagttgat catgttctag ttaaactttt ttaaggctct attccaggtg caaatggttt 72220 72280 72340 72400 72460 72520 72580 72640 72700 72760 72820 72880 72940 73000 73060 73120 73180 73240 73300 73360 73420 73480 73540 73600 73660 73720 73780 73840 73900 73960 74020 74080 74140 74200 74260 74320 74380 74440 74500 74560 74620 74680 74740 561999 78 tccacgttcg tttttagttc attcatttca cggcttctct ctctctattc ctttttttct 74800 ctctgctgac ttttctctgt tctcactggt gactttcatt ttagcgtatg gactttcttt 74860 ttctccccca cattgagaga gcaagttaag tctgggaggc tggagagatg caaagagtta 74920 ttttgcagca gtgaateage agatgcagaa gtagtcttct cctagttctc atttcaggct 74980 ggaggcagac agggagagga agtggcctcg gatttgttgt tgatttgggg aggatgagca 7504 0 tgggggtgga tctgctgatt tctgttctca ttagctttga aaacagaatg cctcctggca 75100 gcggcttctc tgtggcctgc ttttctgctc cggaaactgg gggagggaaa cccctgacag 75160 atgtgggctc atggggcaag tatctgttct gtggggttgg gatgaggggg agaggcaggg 75220 ctttgctcaa gaagaggtgg ccagacagca gctggaggga cagccaggga gggaaggaga 75280 catatttcct ggggaaggaa gtggcccgag cagagacctc agcatgtaga gtctggagag 75340 caggatgtgt cggggtctac aggatcaccc tcaaattgga ggatttgcta gaagaattcc 75400 caggacccag caggtagtca cactcacttc tgtgatgtgt cacggtgaag gacagaaagc 75460 acagtcagta aagggggaag gcacggcggg tgaagtctgc ggggaccagg cacaggctcc 75520 cagagccttc tcctggggca tcacacaggg cacacttcgc tccccgtgtg aggtgtcctc 75580 agccagggac gctcagtcag gactcagcgc ccagggtgtt tattgcgggc accctctgcc 75640 tggcacggac tccagactcc aggaaggaaa gcaggtgtca gcataaacta acctgtttta 75700 caaaccctct aggcccaggg agcccctctt atctgtcagg gagagatggg agccccctga 75760 aatccaggat cccagaggcc aaacaggggc caccatgcca ggagggcagc ctcaggccca 7 5820 ccggatgaac gctttttcac acatggggga tctcactcac atcctgccag gttctcaagt 7 5880 tcaaactctc cccggtcatg gcagcatggc ctttggccag ttcctctgtg ccctggggcc 75940 ccagaagacc tcaggatgct gggctatgct agaagataat gcctgttccc actggtgtct 7 6000 gtgataacga cgcagcgttt atggccctga gtgtggctgt cttgcatccc tgactttgcc 7 60 60 cttgcagaaa ccctacgagg tgggcagagc tgtctccact tttcagatga gaaagagtct 7 6120 cagctgagaa gcagcagtct gtcctcaaac ctagattcct ttccattctc atttcctttc 76180 tttaaaaaaa tttatttggc tgcattgggt cttgggtgca tcacgtggtc cttcatggta 7 624 0 cacagattct ctagttgtgg cacgggctca gtagctgtgg cacgagggtt cagtcacccc 76300 gaggcatgtg ggatcttagt tcccccacca gggatcaaac ccgtgtcgcc tgcattgcaa 7 6360 ggcagtttct taacccctgg accacgagga aagtccctcc agtctcattt cctgacacca 7 6420 ttctggtacc accttcctgg tttttggttc tagatgtgtg tcacttctcc cgttattgac 7 6480 ttactgctta aaaaacaaga tctcattaaa aaaaaaaaat cctgtacaaa ggaagctttc 76540 aaattggaaa ttaaaaattt ctccaggact tcectgacag tccagtggtt aagattctgc 7 6600 gcttctaata caggggcaca ggtttgatcc ctggtcaggg aactaagatc ccaaagccaa 7 6660 aaaaaaaaaa ttctctccaa atggagagtt cactggcagt ccagtggtta ggactccacc 7 6720 cttttgctgt ggtggcctgg ggttcgatct atggtcgggg agctaagatt ctgcaagctg 7 6780 tgcagtggag ccaaaaaaaa aaaaacaaaa ggaaaaagat tttctctaag aaggttaaca 7 6840 taaaattaaa cataaagaaa aagtatcact aaactctctg tgaaatatca tcgcctgccc 7 6900 ttggcatgag ctctgttgaa atagagattc catgaaaatg tatttcagag gcactaggga 76960 ccagccacgt ttacccggtg gctcagtggt aaagaatcct cctgtcaatg caggagaagc 77020 gcgtttgatc cctgggtggg gaagattccc ccggagaagg aaatggcaac ccactccagt 77080 attcttgcct ggaaaatccc atggacagag gaggctggtg ggctatagtc cacggggtca 77140 cagagagtcg gacacgactg agggagtaaa cagtagggac caggcatgct tcctgattca 77200 gactttctct ttcagatgct ggcaggaggg aatggagagg ggtggggggt tggagaagat 77260 agtgacttta tcaacgtgca aatctatttt atttggaggc tggagggaga ggcaatgact 77320 gagaaatatc cttctgtact caggaatgcc ttgtatgagt ggaaagatgg aaaggattat 77380 tttttcccat ccagagatag tctggttctc tggaggggtt ccctcaacgt tgtggacata 77440 agagccattt ggggaatatg tttttaccca tcaggggtta gagtgggtag aggagacccc 77500 agcagatggg cacttcacct gggacatgca ccttcttgaa tgggaccagg gagaggggga 77560 ttagaaggga ttcctggggg ttcaggaagg gctagtccct cactgtgctt gttgccgcag 77 620 gtc acc gga ate ccc atg aac tgc tct gtg aaa ctg cag ctg age ctc 77668 Val Thr Gly He Pro Met Asn Cys Ser Val Lys Leu Gin Leu Ser Leu 380 385 390 ttc gtt aag tct gtg aaa ggc ate gg gtgagtgggg tctgtgagca 77714 Phe Val Lys Ser Val Lys Gly He Gly 395 400 ggggctgcct tgcggttgac agategggga ctcagggaga ctctggcctt gttcttggga 77774 actCaagtct ggggagggat gtgggatttg gtccctgccc cttgaactcc taaggagaat 77834 cttagctctg agetgagtcc agaccacatc ctgacaccct tgccttttct ctgttgacgt 77894 ttgtcccccg ttagtcagaa tattctatcc aagctgtttc tcccttgaca gggtagcaaa 77954 561999 79 aattaaaacc acctgagggg ttgctttgtt tccttgggtt taaagcaggt cccaatctcc aatgtttgtg ccatatataa ctgtctctag agtaatattc acacacacac gcttctgtgt tctctgggta ttaagcacag taattcacac agttggacga ctgtaccatt ccatttctct cttctttcac tcagtccatc caactgatag atctttaaaa aggctttctc gttgcggtgg ggtgtaccgg gggtccccgg gaatatttat ttacagcaga gactgttatc agcatctcca tcagatggga aagatctcct ggacagagag aactttcact nnnnnnnnnn tcgttgaggt agagggcaga tcccgaccag ccgccaacga tgaagtgttt ctggagttcc cgggcaggtg tctctgtaga gcaggtttca aaatgttgcc cacacactgc gggctggggt ccagtgtggg ttctccgagg tgtgcattta gacccccctt taaattcatt ggatttgggc ccagctgtgc tcttgtttcc gaacactgtg actggaatgt tgtctcctga aagccattta ttttcatgta gtgtttgaaa ggagaggtgt tctgccctac tccttcccgt tctcactagt caactgattc tctctgtttc gtgttaatat gcccatccgt caatatatat acacacacag cttggctgca tatacccaag gttttctttt ggtaccaaac catcaecaea agccgtcact ggatttaccc ttagcctggg ttactgctga acatttgggc aagtttgtgt tggttgcagc cttctcttgt cttagttgcc cattggcagg gcctgagtat attgctgggt cagggcggct tgtcttcgcc atgaatcttg ggagtaggaa cctggtgggc cactcattca nnnnnnnnnn gcacaggctt ctcagtagtt ggattgaacc gtccaagctt gtggctggtg ggagggcccc actaggacag gcaccagggg gaatctggat caagccagcg tactttgatc ggtggtctca caaagtctag aacttctgtg attgacatca gtatttctag tcactaccca cttttgtgcc cttctttaag tggcatttaa gactttaacc attgccattt gtctcctgta acaactagac tccacattca atggaaactt ttttttatgt agcagagtga tcaggtcgcc tatctatttt cagctccctt tgcttttcaa acaacatttg gtctctgcaa atatatatat cacatcttta gtgagcactg agtgggcttg tctgtttttt tcacccttta gtccattttg ccccacttcc atttgggcca gttctcaggg ataatatgcc tgtttctgct ctttcgtttt gggcaggggc tgtggaggac ccttggcacg cagattctta gtggatgtat cctctggtgg acactatttc aactcttgct cctgcaatgc atggcaactc tacagtccat ggatagagcc nnnnnnnnnn ctcgttgcag gtggtgtgct tgtgtcgcct tccatcttta agacagcccc ccatcactca aggaaccgga cctcgggctc tttaacacct gtcagtctct tctctggggc aagatggtgt aaacacagct ctgtttggtc gttcttagaa aatgttttcc gcactctcct gactctagtg cagactcagg cagctgcatg ctccaggttc cctcttccag ctggcaggca aacagtaata gtcctcacta ggccaacctc ttttaattgc ctcagctctg atggagcact acacatagta ccttccttgg atagattcat tgtttctctt actgcacaat atatatatat tccattcttc gggtgcatgt ctgggtcata tgtttttcca agaacataca aaacttttcc ctcagcctca tcttatctga ctcatccatg attgcctggc ctttggccat tggctgtgct tactctctgg aggctctagg tcaagtcttc accactggac gttttcattt ccctatgttt acactcccac ataacatcac tggagacccg actccagtat gggatggcaa gggtgaggga nnnnnnnnnn tcacttagct ggcttagttg gctttggccg ccttttgagt atgttgggcc atgcctggtc caggctgggt agtgtggeca cagtttcacc ctgggtggct tctgtgtccc ttatcaacat gttccagaaa acaagataga actcagatga atgtctgtct taataagtga cctgtccccc aaggtgaagt ctcagtcgct ctctgtccat gggatcttcc aattctttac aacactgaac gaaccctgtg attctctagt agtgtagttg tgtgtgcatt gagtacagtt tccattgtat aatccatacg ctgtcccatc tcttatttca ttcattcctt atacacacac tattgacgaa atctttttga tggtagacag gtaacagctt gtgcagtagg atcaacccac ggcaaccctt acagagttgt ttgtagcagg aagaccgtgc tatgaataat acgtctttat ttgcgttgca ctcacaggct ctggaccagg caccagagaa ctcttgggta aaccgtttga cagcagcgtg tatagcacag ggttcgatcc tctggcctgg agagttggac gcatgctgat nntaagtctt tgttgtagag ctcgacagca gtggattctt ctgctcagag ttaggcgtct tgctaacttc ggggacgggc catccgatgc tgttgagagt ggattctact cagctgttca ccctgagggc cagagccatc ggcacgaggg aagaggccaa cctctgcagg tctttgagaa tagtgggttc tacggaatat aggtcatgtc gcagttttcc caacccaagg catggaacca actttctata gggaattcat gagaccattc ccttacgatg catcccctct ccctgcgctg ttatgtcaat tttgttctct tttttagatt ttctgtgtga tttatgactg acacacacac catttgggtt attacggttt gagaaatttt catcaagatt atattcacag agaagaaacc aatttactta atgtgactgg tgtcagcact ttttattcat gctgctctaa tactgtgttc cagacttctc cagtagttgg gatgggacct gtcctgctct gatagatatc ggaaccgtca ggagggtgcc agctggtggc ctgggttagg agaattccat acaactgagc tgnnnnnnnn cgttgctgtc ttcgggctct agtggaatgt aaccactgga agtttgttcc gctgctcagg atgctcaggg caagggccag atttgcaaac tcattctgac tgttggtggc gagctgctgt tgttgaggaa aagctgtgac ttgcacggtc agtgaaggtg tgatactgtg ttgtgattga catctcccct ttgtttgttc tgactcttta aggcaagagt gttaaacctg cccacctgga tgctgggtat tataatcccc 78014 78074 78134 78194 78254 78314 78374 78434 78494 78554 78614 78674 78734 78794 78854 78914 78974 79034 79094 79154 79214 79274 79334 79394 79454 7 9514 79574 79634 79694 79754 79814 79874 79934 79994 80054 80114 80174 80234 80294 80354 80414 80474 80534 80594 80654 80714 80774 80834 80894 80954 81014 81074 81134 81194 81254 81314 81374 81434 81494 81554 561999 80 atttttcaga taactgactg ctcggctgtg cacagggtga acataggaca cagggatgcc gctttctgcc taaaccacat tcaaagagga tgaagttttc ccattcctca cctgaggcac ggaagcacag tgtcgggggt atttattata agtctggggg tgtttcctcc cagtagaaac tggtatgcac tgtttgttag agaaatgcac atttaaaaag agaccacgcc cctgacaaaa agagggtaag tcccaggacc tagaaaatat aaccagacag tcccagtgat tcttcccagg ccaaattcca ctgggtacat atcagctagg atattgtcag gatatagtcg cttctcactt gggcatgccc aetetcaggt tcaaaacata aggcttctag gagttgtgat ggatgtttac gactccctga tgccatcctg tccctggcag attgtcaggt ttgtagaatc gcttgatctc gaggtcacac tcgatgattc accagcaaag agctctctct atgtgtgaag tgggggctgg aggaaagcag caaaaaactg cacctaccac tgcttcccgt acaggtccag ccttatttcc agcttgttgg actgggaaga gggacaggct taggggagtc tgtcccctac tcacatcggc gggatcagca gcattctgtt atagaagaca caggagatac agctgtctga ag a caa Gin 81614 81674 81734 81794 81854 81914 81974 82034 82094 82154 82214 82270 act ggg aac ate cag cca gtg gtc ctg ccg eta atg tgg ttc gaa gag Thr Gly Asn lie Gin Pro Val Val Leu Pro Leu Met Trp Phe Glu Glu 405 410 415 82318 gtaaggctgc ctccctctcc ctgggtatca ggtacttgtg gtcatgtgtc tgatgcctca 82378 gtgaggagct gatgtgggca tttgaggcaa gaggctgtgt gtggtgggga ggcgggactg 82438 ggtggctgga ctctgtgatg cagaacagtc tattagaaat ttcgccctct ccaagcacct 82498 tatgtcaatc acgccactgg actgaaaggc tcacttgaga tgagaaagac gaagctgcct 82558 gagattgtaa agtccctggt cagggtataa aataattttg etaatgeatg tgaccacttc 82 618 tccaaagatg aagggaagtt cgttttcagt gtgtcagctt tttattgaac ttccttttct 82 678 cccagctttt caagattcct tctggcttag ttcattttaa ttcagggtga gctaacagcc 82738 ggaacagaca gtctccaaat gtcaaattcg cttcctgctc acatcacagt ctgatggagg 82798 ggggaggtgg agctctgctc catgcagaca tttggggaca tctgggctcc ttccatcgtt 82858 tggtcctgac ettteggaga tcctcagaat ccatttccat tcagtaaatt ggcaaagaga 82 918 tggagagaat cacatgggag ccaggcgtgg aaattgtaac atccttgtgc ccacatccca 82 978 ttggccagaa gaagtcacat gggcctgcct agetgeaagg gagtctggga aatatagtcc 83038 tgttgtgtgg ccctggagga catagaggct ggctaatggc atgctttggc cagtggtcat 83098 gggatgggga agggatagag tggcatctgc accctcagct tatagcatgt ggccctagtc 83158 gatctgtttg tccttcttcc ccag agt gga gca atg gag ggt gag acc ctt 83209 Ser Gly Ala Met Glu Gly Glu Thr Leu 420 425 gag acg ttc tat ate cag ctg gtg ttg atg ccc aaa gtg eta cac tat Glu Thr Phe Tyr lie Gin Leu Val Leu Met Pro Lys Val Leu His Tyr 430 435 440 83257 gcc cag tat gtg ctc ctt gcc ctg ggc tgc gtc eta ctg ctc ate ccc Ala Gin Tyr Val Leu Leu Ala Leu Gly Cys Val Leu Leu Leu lie Pro 445 450 455 83305 ate ate tac caa ate egg age caa gtaagtagtg gtcagagcat ggcctgggct lie lie Tyr Gin lie Arg Ser Gin 460 465 83359 gagtggtccc tcccttcctg gcagtcctag gcaccttctg caccgcagtg agetgetgge tgttgatggg taaacagaag cagtgctgtc taatagccac atatccaaaa ttgcttgcct ctgtgcccat gtctctactt ttaccacttc gttttggatc tgccaggttc catgcgggga aagtgatttc caggacagat gttaggtaaa gattaccatt ccttggggga ggacacagcc gggctgggga tctgcctgtt atgagacctg tggcaggtac ccagcattct accaatgata accacacgag agggaattaa tcaatctgca aggaggggct tgcaccctca gccatccatc ctgggaggct gtactcgaat agcgcgtaca agttggaaag aagatgatag ccacatgtgt tttcaatcat atcaatataa taaatcagat gcctgcaccc actgcatcct agggggccca ggtttgttca acagtgaaca acaggtggga agaatgtgat gattttaaat atcttttatt aaaaattaag tccccaactg acttcgagca tgcctttcct actttagagg tctagtcagc agacagttcc gtcaagtaac gggctcaggg ttaaatgttc taacctggat tacttttgca 83419 83479 83539 83599 83659 83719 83779 83839 83899 83959 84019 561999 81 ttatgttttt gagtgcagtc tttggaatcg gtgtgtattt gacacttaca gcagatcttg 84079 attgggactc accccatttc aaagcctcca cagccacatt tggctgctag actgggcgcc 84139 acagtatcca gggaggttgg ggacagtgat agcaataatg tcagaagagg tttctctgaa 84199 gaggttgaat ttaggcttag atgtgaatgc tcagcacaga gtcatggaaa gactgctgca 84259 ggtggaggaa tggccagtgc aaaggccctg aggcaggaat gggtggggct gtattggagg 84319 atcctgccac actagattgg ctgtaggcgc agcaggggag gtggtatctc agaggcagag 84379 gtaggtcatt cctggccctg taactcaggg tgagcggctt ggaactgatt ccaagggatg 84439 actggaagct tttaactcat tattgactgg ggaatttttg cagaaacaaa tgcctgtagc 84499 cggcgatttt tattttggcc agccaaaaat taattctgtt atttgactgt gtgtaggtta 84559 ttgcaatcaa tagttacccc tgacacacct gtacagtctt ctcattttca tgaaatgttg 84619 tcttcaaccc actcctctgt agaaacaaag gagcattctc tagcatcgtg agttctgttt 84 679 tcactttctg catcaaaatc aatcactaag gttttttgtc tctttgttgg tctaatattc 84739 acattgtctg aatcagaact atgttctgtc ttctgagaca ctagtatata tgttgtgcag 84799 acagtcagag aaagtatctg cctaaaattc accaaaaaat tattcttcac tcagaatttc 84859 acaatgtgtc attttgggga aaattttacg tttataatat aaacaggatt ggaacaaaaa 84919 ccttaatgaa gcaaaccgtg agtgataatg acaatcataa gctgtataat gagcccttaa 84979 tcaattttaa gctctaacaa cttcgcatgg tgatgttaat tgtattaacg tgtttttttt 85039 ttttttaaac atgttaattc actctctaaa aacatgttga tacgtctctg gtcaataata 85099 ggtttaagga gaggagtgac tgggtcggtt gacatgtggc aactattaat agaccttcca 85159 atgagaaagc agaagcaggg ggaccagtgg ggaggcccag gtgagctagg atactctggc 85219 ttggaccaga atgggagcaa gtattcagga acaagagccc tgaagaacca gtcctggggc 8527 9 atctgccatt caaagtctgg aagaagatca ggggacaaca aataccccct ttcttttttt 85339 cttttctttc ttccttggtc tttttttccc tctgccttgc ggcagggcat gcaggctctt 85399 agttccccaa ccagggattg aacctctgcc ccctgcagtg gaagcacata gtcttaacct 85459 ctggactgcc atggaagtcc caaggaaaac ccctttctga tttggcatca trmnnnnnnn 85519 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn naagttttcc tggagcccca 8 557 9 ggcctgggcc tcggaggggg gttgcccagc ttctctgctg ccccctcggg ccacctgcaa 85639 cacccgcttt cttgctttag ctttggcaaa cagccttttc tcttttgcct ttttctcgga 85699 ggcagattgc atgcttgctt gcttgttggt agctggggcg atattaaacc tttccaggct 85759 cttaacctgg gctttcagac ctttatcacc atttctcttc caaattcccc atccttcggg 85819 ggaaaaggtt tgcactgatg tgagggtacc agggcctctg gtgtcttgag cccccaatga 85879 acgttgggga ccctgtcctc tgtcactgtc ccagccagag cagaagggaa ctcccagtgg 85939 tgtttggaat gaattatgct ggaggacaga ggggaccaga ggccctagcc cccagtctgt 85999 ggatgactcc ccaggcccag catcaccgtg cgtttccagg agtgaagaca tacacagcca 86059 cacaggcggg gggtgggggt gaggtggacg gggccagggg aagcagctgg tgagggcggg 8 6119 gccagacagc ccccgccctc tgtcccctct gtctctctcc taatggtcta ttagaagggg 86179 aagggggagg agactgagga tgcaggatgt gaacacttcc ctgcttcacc ccccatcctt 86239 gttccccaag tcccataaga ttattcatcc tttctattaa ttatggtgag gctccctccc 86299 cacaggttat agtggaggct caatcaatga aatttatccc attgtgtgtt ttggacagag 86359 gagcctggca ggctacagtc tatgaggtcg caaagagctg gacacagctg aatgacaaac 86419 acgtgtaatt tttggtttag caagggttag gccttttgtg tttttatata tttcccctgg 86479 gggaaaaaaa aaagaatggc acgtcgttaa actcaacata ttttaatttc tcttcaaaga 86539 cgtttttttt gttcaagtca tttttgaaaa aaaaattgtt ttaaacagaa acacctctgt 86599 ttctggtcta tttaaaatgc cacctgctgg ctcagtcagt acagcatgaa attctccaca 86659 tgccacttgc tcacctctct gagactaggc cgagccgcca tgagcaccat agcccatctg 86719 ccctgctcaa cttggcctaa attctcaggc cgaaagaccc tttttagttt gcccatctga 8677 9 ccaaatgtgt ttcctgaaga tgctggtgta cgtgtcttgg gtgaagggca ctatcacttc 86839 ccagatctcc caactgcaaa tcattaatgg tggttggacg tgaaagttct gtaccccacc 86899 ctctgctttt tgaagaaccc tgcaaattga agaaacctat aaaacccgtg tgtgttcagc 86959 tgcgacggca actataaatt agcttggtta tcttctag gag aaa tgc tat tta ttt 87015 Glu Lys Cys Tyr Leu Phe 470 tgg att agt ttt aaa aag ggc tea aag gat aag gag gcc gtt cag gcc Trp lie Ser Phe Lys Lys Gly Ser Lys Asp Lys Glu Ala Val Gin Ala 475 480 485 87063 tat tct gaa ttc ctg atg aca tea get ccc aag gga act gta ctg caa Tyr Ser Glu Phe Leu Met Thr Ser Ala Pro Lys Gly Thr Val Leu Gin 490 495 500 505 87111 561999 82 gaa gca aga ctg taggtgggta ccaggtaatg cccccccact ccccacctcc Glu Ala Arg Leu 87163 tcccgtagtg cctgcacttc gtgatcccag gtttgtctta attgactctt ctgaaataga tggtaaagaa cccctgaaga agaggagcct cacgaaccaa ggagtgtagg tggagtctta tgggatgact aggactcagt agaaataaca attttttgcc cgttggttac geagaagaag tctgaggtct tactgttggg ctgttatttt cgggtttaaa ggggccagtt gaacacgcca agcagaaggg tggggataag tgggagcagg tcctctggga caaaattccc tagaagggtg gtggctatgc cctccaaaaa aagcttcaag cacagcatgt agtatggtct ttgccttgcc tggggaaggc gctagtggtc tgggggagat cagaaagatg tttaggactg cctttgtaca gatctttctt acagtgttag tttgttacat attggaagct cccataggtg atctctcagg gactggcgat tcccaccctt tgctgtcttg tatactgtat catccacctc gatccacatt atgtgggagt agtagaactc acctgtctac atgtggtagg aaaaggcagg ctggggaagt atcatggaca tccgcctgcc aggaaatggc ggtgggctac ggggattatg atcttagttc gccactggac gaccaaggaa ctgaaggtag gcagcaaaga taagtgtgtc ctgaaattca taggctggtg taggagagct gtttgggcag ggtttctgaa tagctttctt ggctgctcca agtgctggcg agggacagct agagcccgac acatcctagc cgccttctta ttctgcagaa ttactggctt ctccctggac aaatccacac ttatcaggga gggatcttag tatggaccac ttctggaagg agtgtcctgt cagaagacct cccacacctt tctttatgtg aaggtttaga gtgatcaacc accttcttat ggaacaggag gtgtctttct aattacttta tacatgtgtt gtcatcccag ctgtttcaca gccttctccc catatagggt tggtgttttt attagaactg gctgagaatc gtgggtcagc tggcaccctt agactgaaaa ggatggaggg atcgaggttt ctgctaaaaa ggccccggga agtgcaggag atcctgctcc cgtccatggg agtagattgg cccaaccaga tgccagggaa gaaacaattt gattgccaga gatgctcagt ccatgcaatt catttaacga ccttgtgggt aagtccagcc gagtgagtca aaatgtgccg cagagctaag gtttgcttcg tgggtgtgtt gtgtccccag cacgtcaggg cagagatggg ggtacagttt acttgtttcg ttagcaaaga gcagccagga caaacattgg aggctttttt ttccctgacc cagggaagtc ctgagcccag tcatgctgtt agaatccett gccttgcact agtaagaaaa acacagaata tggacaactg gagtctcatt gaggaggagg tgaaaaagca cagtgttgtg ccccatcctg tgcaccagct tatgatagta acagagtcca catcgttacc ctttctgact atcacttttg tgccctccac ggggggcact aaaggctttg tgtgtacttt cgattagtag aggtcagctg tgaagatctg atgtgttatt acgtgggttt agcattcttg gccgcaaaga tctatttatt aatcgaaccc gtccctgata agacatcacc ttgagcaaaa taaatgtgaa gtcgggacac gctgccctcc tcaccttcct aggggaggtg gtcgcactat ccctcgccag cctgaggacg actgccaggg tgctggcagg ccagattctg ctgagtgtta taccccgctt ggccccttgg tgttctgggg tagagctggt tatcctgccc agtctcctct tttttttttt aggaatcgaa ccagagaagt atgagacccc ttagggggcc cagggaagcc tgggcatggg atacagcccc caacccatgt tactaggcag atttccaagt aatctgtctc gtttcccctt gtgggttttg accctccctc ccgagcaccc tacatgtttc acagtctgtt atctttctaa tactteactc agcgaggctg ccaccacccc ggtcttgttc gatctcatcc gctgtcttct ggctggtgtc cttctcagac aggacattct ttaatcccct gatccctggg cctgggaaaa gttagacacg tatttttggc acgtccctgt tagattggtt tgggcaccca acaaaccaaa ttttaggtaa gcttatattg ctgtatctga ctgggctgtg acaggcagag ggttgtgcct cttcttgggc agtcgacagc ttggaggagg cgctgccagt gggggctcct agggggtgtc tccttcctct atacccctcc gtgtaagggt gattgtgagg accactccct tgactttacc ttaatctttt cctgcgcccc ttttgattaa tatccccagt aggcaggcac tctaaccttt gtggccttta tctgcgcaga atagccccct ccctgcaggg cctcattcct aggttctgct ttcacttaaa ccatacattc ccacctccct tgcctcatgc agtgctattc ctttacatct attacatata tgtataatag tctccagtct gactgtcgcg tgacaccata atctcttcag tacattctga ttattttcaa ttccggtgca tctaaacctc ggtgactcag ttgggaagat ccccatggac actaagcgca tgcgccctgc attggaggcg taagtgaatc gtatatatcc aaaactccca acagtgaatg aaaacctgtt caacctacct ttcctttagg ctcttagccc ctgatcatag tgtgcctgcc ccttctcttt agagaaaacg ccctgcgagg cctgccgatc ctcctgagct ctcccagaat cccttccagc gaggtcttcc tctcagccct gccccgcccc aacagaattc gcccatacca ctacattggc agaggttcta catgtgtgct ctgagaagtt tttgaacacc gctgctagcc catggcctgc tggctgagtg catgttcctg cagttaaagg tccttgaggg aaaaaaatta acatgaatca ccccaatccc attgaacctg tctcaaatca gtgtctcttt tatgggttaa gctccagttt tagttctttg tgctgcctgt cccccaccac 87223 87283 87343 87403 87463 87523 87583 87643 87703 87763 87823 87883 87943 88003 88063 88123 88183 88243 88303 88363 88423 88483 88543 88603 88663 88723 88783 88843 88903 88963 89023 89083 89143 89203 89263 89323 89383 89443 89503 89563 89623 89683 89743 89803 89863 89923 89983 90043 90103 90163 90223 90283 90343 90403 904 63 90523 90583 90643 90703 90763 90823 90883 90943 91003 91063 91123 91183 91243 91303 91363 91423 91463 91543 91603 91663 91723 91783 91843 91903 91963 92023 92083 92143 92203 92263 92323 92383 92443 92503 92563 92623 92683 92743 92803 92863 92923 92944 561999 ctcccctcct caggagagga ttccccaccc ggttttgggg aggcgaatga gcttaccttt atctgctgta ttccttccca ctgtgttatg gtcctggtgt gcatctgctt ttctacatac ataaccagct aattaaaaaa cagcattaat accccaccaa tgtgtgcctg atctctggag tgtttgtgac tgatatcagt aagccctgaa gtttccccat gacccttgca ttgattggtg tgaaggggcg tagtgatgca ctgtctggcc gctccgtgga cacgcacgcg acaggtgtgt gtcaacaagg ctatgggcac gcccgctcca gcatttgccc tgcctgaacc ccactggaca atgggtttgg agctggtcac gtctgcagct ggtgtctggt tcttttctac ataaaccttt tgctcctggc gccgggagtt cgcagtgaag gttttccagc agatagaagg aatttttttt caaccaagtg cacccccatc cagcagcttc cccagctcgg ctctattctt atgcgttcat ctaggtccat aaaagtaaag tgttcacatc gccccacagc ttttcccctt gacatgctat agctgtgtag ggacgtttag taccttgctc ctctaaaatg gttgcggccc gggactgctg gggtgggttg gcgtgccctg actcagccag cagcccacca cacacacaca gcagatactc gctgttctgg ggggtcccct tctgtgaaac tgagaggccg tggtgggcaa gccatctggg gtgtgcgggt ttcctgactg accaggtgcc caggagaccc tggaagaaaa ttaaaaagtg tggaatgtgg tcaccttccc cagacctctg cacagctgct cttaggggga tttttgttgg actcatctat tcacccgtct ccactggcta cccgccctcc gccctgcaaa atacgatatt ccatgtctct ctagatgaac ttgtcccagt ccctccccct ctaaatctgt gtgaatcact gttgactacc gcggtttcca tgcccttttc gggtgagggc aggagtctgt atggggactc tgggcggggc ccttttctcc accaacttcc gtcccaaagt cacacacaca agggatggag aaacttctct agcccaggct actgcagccc ccactgtccc ggttttgtgg cctcagaaac cccttccctc gggtggcagg agggcagagg atctgtctgg actgaatatc a 83 ctcagtgtgg cttggtcctg ggctttgtgc gggcaagaag tgaaaagtta agtatgattg atgcatgcat aggtcagcac tctgttttac ccttcccctg tacgttcatc tgtttttctc ttaatttcat gacaccgagt tctccatttc atcccaagac atccatgagc ctgcagctgg actttgcgac attttcctgt tctgtattag agggcgggat aggtcactcc ccaggggggt ttggcatggg acagggttcc cagcccctac ctgagccccc catgcccctg ccgctgctga ccaagtaatc cttagtgact cttcctggtg caaatccagc ttcagcaccc ggagatgcat cagcctaaac aagctgtgcc tagagaggct gactcctggt atcttttaaa gtagcctgag cccagagtgg gcaaggtcca gcatctctct ccccagtttt ctttactatg atatcccctc agagccccga atatggcagt ggtccgcatg tgttccattt tctgacttac gaaatttcct tccctctcac actgtgtgcc agtcatttct aacagatcag ctttcccttt tctatgtttt gtcgacagta gcgagtccct ggtgtttcat agtcttttct ttccattctg cggggcttgg aaagatgcca accccgcttc cacctcctgc gcacgtgtcc gagacttggg cacaggcctg cacgtcacct gctcatccat ctaggagtgc agcccggacc cctaaacccc tgacatcatc ctgagctgcc ccgtctggga ctctgggaca aataattcac atcagtccca agtttgattt agcgggcagg ccattcttca tattatcaca ttgtgccagt cctcctgagc actgagctct gcccacatcg tctgctccct tttttctaga ttcactctgt taaattgtga ccagactcag ctttgccccc aggaatttgg tttttgaaac ctcgattttc tgatgeatta ttgcttatat ttggacctca gagatgggga catctcaggc gtggaggatg tggccggcct tcagccagcc ttcaggactc acattgcacg acacgtgtgc ggaaagactg accgtaggtg ctccccagcg gcaggacagg tgacttcttg aaagctgtag ctgcaggatc ctgtatactg ccccaccaag cgtcagcctg agcctctgtc aattgaagta <210> 2 <211> 509 <212> PRT <213> Bos taurus <4 0 0> 2 Met Gly Asn Leu Ser Arg Ala Arg Arg Val Thr Ala Ala Leu Gly Phe 15 10 15 lie Gly Leu Leu Phe Ala Val Leu Gly lie He Met He Val Met Val 20 25 30 Pro Ser lie lie Lys Gin Gin Val Leu Lys Asn Val Arg lie Asp Pro 35 40 45 Asn Ser Leu Ser Phe Asn Met Trp Lys Glu lie Pro Val Pro Phe Tyr 50 55 60 561999 Leu Ser 65 Gly Gin Phe Arg Phe Leu Gin Glu 130 Ser Met 145 Leu Ala Gly Glu Gin Tyr Ala Glu 210 Val Lys 225 Ser Lys Thr Ser Glu Phe Glu Gin 290 Ser Thr 305 Cys Pro Asn Ala Val Leu Ser Leu 370 Val Tyr Lys Pro His Lys 100 Glu Tyr 115 Ser Asp Met Met Phe Ser lie Met 180 Phe Pro 195 Leu Asn Asn Phe Val Asn Gly Gin 260 Tyr Ser 275 Gly Val Leu Phe Cys Arg Pro Leu 340 Ala Glu 355 Phe Leu Phe Phe 70 Gin Val 85 Ser Asn Arg Ser Tyr lie Glu Asn 150 Thr Leu 165 Trp Gly Asn Ser Asn Ser Ser Arg 230 Tyr Trp 245 Met Trp Pro Glu Phe Gly Ala Asn 310 Glu Ser 325 Phe Leu Ala Val Asp lie Asn Lie Gin Glu lie Thr Tyr Gin 120 Val Met 135 Arg Pro Gly Gin Tyr Asp Leu Pro 200 Asp Ser 215 lie His His Ser Ala Pro Ala Cys 280 Gly lie 295 Gly Ser Gly lie Ser His Ser Gly 360 His Pro 375 84 Val Asn His Gly 90 Phe Asn 105 Phe Gin Pro Asn Gly Leu Arg Ala 170 Asp Pro 185 Phe Lys Gly Leu Leu Val Asp Gin 250 Phe Met 265 Arg Ser Pro Thr Val Tyr Gin Asn 330 Pro His 345 Leu His Val Thr Pro Glu 75 Pro Tyr Asn Asn Pro Asp lie Leu 140 Leu Lys 155 Phe Met Leu lie Gly Lys Phe Thr 220 Asp Lys 235 Cys Asn Thr Pro Met Lys Phe Arg 300 Pro Pro 315 Val Ser Phe Tyr Pro Asn Gly lie 380 Gly lie Val Tyr Asp Thr 110 Lys Ser 125 Val Leu Leu Met Asn Arg His Leu 190 Phe Gly 205 Val Phe Trp Asn Met lie Glu Ser 270 Leu Val 285 Phe Val Asn Glu Thr Cys Asn Ala 350 Pro Lys 365 Pro Met lie Gin 80 Arg Glu 95 Val Ser Arg Gly Ser Ala Met Thr 160 Thr Val 175 lie Asn Leu Phe Thr Gly Gly Val 240 Asn Gly 255 Ser Leu Tyr Lys Ala Pro Gly Phe 320 Arg Phe 335 Asp Pro Glu His Asn Cys 561999 85 Ser Val Lys Leu Gin Leu Ser 385 390 Leu Phe Val Lys Ser Val Lys Gly lie 395 400 Gly Gin Thr Gly Asn lie Gin 405 Pro Val Val Leu Pro Leu 410 Met Trp Phe 415 Glu Glu Ser Gly Ala Met Glu 420 Gly Glu Thr Leu Glu Thr Phe Tyr lie 425 430 Gin Leu Val Leu Met Pro Lys 435 Leu Ala Leu Gly Cys Val Leu 450 455 Val Leu His Tyr Ala Gin Tyr Val Leu 440 445 Leu Leu lie Pro lie lie Tyr Gin He 460 Arg Ser Gin Glu Lys Cys Tyr 465 470 Leu Phe Trp lie Ser Phe Lys Lys Gly 475 460 Ser Lys Asp Lys Glu Ala Val 485 Gin Ala Tyr Ser Glu Phe Leu Met Thr 490 495 Ser Ala Pro Lys Gly Thr Val 500 Leu Gin Glu Ala Arg Leu 505 <210> 3 <211> 1530 <212> DNA <213> Bos taurus <221> CDS <222> (1) .. (1530) <400> 3 atg ggc aac ctc tct Met Gly Asn Leu Ser 1 5 aga gcg cgc egg gtg acc gcg gcg ctg ggt ttc Arg Ala Arg Arg Val Thr Ala Ala Leu Gly Phe 10 15 48 ate ggg ctc ctc ttc gcc gtg lie Gly Leu Leu Phe Ala Val ctg ggc att ate atg ate gtg atg gtg Leu Gly lie lie Met lie Val Met Val 25 30 96 ccc tea ate ate aag cag cag Pro Ser lie lie Lys Gin Gin 35 gtc ctc aag aat gtg cgc ate gac ccc Val Leu Lys Asn Val Arg lie Asp Pro 40 45 144 aac age ctg tct ttc aac atg Asn Ser Leu Ser Phe Asn Met 50 55 tgg aag gaa ate cct gtc ccc ttc tac Trp Lys Glu lie Pro Val Pro Phe Tyr 60 192 ctc tct gtc tac ttt ttc aac Leu Ser Val Tyr Phe Phe Asn 65 70 ate gtc aac cct gag ggg ate ate caa lie Val Asn Pro Glu Gly lie lie Gin 75 80 240 ggc cag aag cca cag gtg cag Gly Gin Lys Pro Gin Val Gin 85 gag cac ggg cct tat gtg tac agg gag Glu His Gly Pro Tyr Val Tyr Arg Glu 90 95 288 ttc agg cac aag age aac ate Phe Arg His Lys Ser Asn lie 100 acc ttc aac aac aat gac acg gtg tcc Thr Phe Asn Asn Asn Asp Thr Val Ser 105 110 336 ttc ctg gag tac cgc age tac cag ttc cag ccg gat aag tcc cgc ggc 384 561999 86 Phe Leu Glu Tyr Arg Ser Tyr Gin Phe Gin Pro Asp Lys Ser Arg Gly 115 120 125 cag gag age gac tac ate gtc atg ccc aac ate ctg gtc ttg agt gcg 432 Gin Glu Ser Asp Tyr lie Val Met Pro Asn lie Leu Val Leu Ser Ala 130 135 140 tea atg atg atg gag aac agg ccc ggg ctc ttg aag ctg atg atg acc 480 Ser Met Met Met Glu Asn Arg Pro Gly Leu Leu Lys Leu Met Met Thr 145 150 155 160 ttg gcg ttc age acg ctt ggc cag cgt gee ttc atg aac cgc act gtt 528 Leu Ala Phe Ser Thr Leu Gly Gin Arg Ala Phe Met Asn Arg Thr Val 165 170 175 ggc gag ate atg tgg ggc tat gat gac ccc tta ate cac ctc ate aat 57 6 Gly Glu He Met Trp Gly Tyr Asp Asp Pro Leu lie His Leu lie Asn 180 185 190 cag tac ttc cca aac teg ctc ccc ttc aag ggc aag ttc ggc tta ttt 624 Gin Tyr Phe Pro Asn Ser Leu Pro Phe Lys Gly Lys Phe Gly Leu Phe 195 200 205 get gag ctc aac aac tcc gat tct ggc ctc ttc acc gtg ttc aca ggg 672 Ala Glu Leu Asn Asn Ser Asp Ser Gly Leu Phe Thr Val Phe Thr Gly 210 215 220 gtc aaa aac ttc age agg ate cat ctc gtg gac aag tgg aat gga gtc 720 Val Lys Asn Phe Ser Arg lie His Leu Val Asp Lys Trp Asn Gly Val 225 230 235 240 age aag gtc aac tat tgg cat tec gat cag tgc aac atg att aac gga 768 Ser Lys Val Asn Tyr Trp His Ser Asp Gin Cys Asn Met lie Asn Gly 245 250 255 act tcc ggg cag atg tgg gca cca ttt atg acc cct gaa tcc teg ctg 816 Thr Ser Gly Gin Met Trp Ala Pro Phe Met Thr Pro Glu Ser Ser Leu 260 265 270 gaa ttt tac age cct gag gcc tgc egg tcc atg aag ctc gtc tac aag 8 64 Glu Phe Tyr Ser Pro Glu Ala Cys Arg Ser Met Lys Leu Val Tyr Lys 275 280 285 gag cag ggg gtg ttt gga ggc ate ccc acc ttt cgc ttc gtg get ccc 912 Glu Gin Gly Val Phe Gly Gly lie Pro Thr Phe Arg Phe Val Ala Pro 290 295 300 age acc tta ttt gcc aac ggg tcc gtc tac ccg ccc aac gag ggc ttc 960 Ser Thr Leu Phe Ala Asn Gly Ser Val Tyr Pro Pro Asn Glu Gly Phe 305 310 315 320 tgc ccc tgc egg gag tcc gga ate caa aac gtc age acc tgc agg ttc 1008 Cys Pro Cys Arg Glu Ser Gly He Gin Asn Val Ser Thr Cys Arg Phe 325 330 335 aat gcg ccc ttg ttt ctc tcc cat cct cac ttc tac aac get gac cca 1056 Asn Ala Pro Leu Phe Leu Ser His Pro His Phe Tyr Asn Ala Asp Pro 340 345 350 gtc ctg gca gag gca gtg tct ggc ctc cat cct aac cca aaa gaa cat 1104 561999 87 Val Leu Ala Glu Ala Val Ser Gly Leu His Pro Asn Pro Lys Glu His 355 360 365 tcc ttg ttc ctg gac ate cac ccg gtc acc gga ate ccc atg aac tgc 1152 Ser Leu Phe Leu Asp lie His Pro Val Thr Gly lie Pro Met Asn Cys 370 375 380 tct gtg aaa ctg cag ctg age ctc ttc gtt aag tct gtg aaa ggc ate 1200 Ser Val Lys Leu Gin Leu Ser Leu Phe Val Lys Ser Val Lys Gly lie 385 390 395 400 gga caa act ggg aac ate cag cca gtg gtc ctg ccg eta atg tgg ttc 1248 Gly Gin Thr Gly Asn lie Gin Pro Val Val Leu Pro Leu Met Trp Phe 405 410 415 gaa gag agt gga gca atg gag ggt gag acc ctt gag acg ttc tat ate 1296 Glu Glu Ser Gly Ala Met Glu Gly Glu Thr Leu Glu Thr Phe Tyr lie 420 425 430 cag ctg gtg ttg atg ccc aaa gtg eta cac tat gcc cag tat gtg ctc 1344 Gin Leu Val Leu Met Pro Lys Val Leu His Tyr Ala Gin Tyr Val Leu 435 440 445 ctt gcc ctg ggc tgc gtc eta ctg ctc ate ccc ate ate tac caa ate 1392 Leu Ala Leu Gly Cys Val Leu Leu Leu lie Pro lie lie Tyr Gin He 450 455 460 egg age caa gag aaa tgc tat tta ttt tgg att agt ttt aaa aag ggc 1440 Arg Ser Gin Glu Lys Cys Tyr Leu Phe Trp He Ser Phe Lys Lys Gly 465 470 475 480 tea aag gat aag gag gcc gtt cag gcc tat tct gaa ttc ctg atg aca 1488 Ser Lys Asp Lys Glu Ala Val Gin Ala Tyr Ser Glu Phe Leu Met Thr 485 490 495 tea get ccc aag gga act gta ctg caa gaa gca aga ctg tag 1530 Ser Ala Pro Lys Gly Thr Val Leu Gin Glu Ala Arg Leu 500 505 <21Q> 4 <211> 509 <2l2> PRT <213> Bos taurus <4 00> 4 Met Gly Asn Leu Ser Arg Ala Arg Arg Val Thr Ala Ala Leu Gly Phe 10 15 lie Gly Leu Leu Phe Ala Val Leu Gly lie lie Met lie Val Met Val 20 25 30 Pro Ser lie lie Lys Gin Gin Val Leu Lys Asn Val Arg lie Asp Pro 35 40 45 Asn Ser Leu Ser Phe Asn Met Trp Lys Glu lie Pro Val Pro Phe Tyr 50 55 60 Leu Ser Val Tyr Phe Phe Asn lie Val Asn Pro Glu Gly lie lie Gin 65 70 7 5 80 Gly Gin Lys Pro Gin Val Gin Glu His Gly Pro Tyr Val Tyr Arg Glu 561999 85 90 95 Phe Arg His Lys Ser Asn He Thr Phe Asn Asn Asn Asp Thr Val Ser 100 105 110 Phe Leu Glu Tyr Arg Ser Tyr Gin Phe Gin Pro Asp Lys Ser Arg Gly 115 120 125 Gin Glu Ser Asp Tyr lie Val Met Pro Asn lie Leu Val Leu Ser Ala 130 135 140 Ser Met Met Met Glu Asn Arg Pro Gly Leu Leu Lys Leu Met Met Thr 145 150 155 160 Leu Ala Phe Ser Thr Leu Gly Gin Arg Ala Phe Met Asn Arg Thr Val 165 170 175 Gly Glu lie Met Trp Gly Tyr Asp Asp Pro Leu lie His Leu lie Asn 180 185 190 Gin Tyr Phe Pro Asn Ser Leu Pro Phe Lys Gly Lys Phe Gly Leu Phe 195 200 205 Ala Glu Leu Asn Asn Ser Asp Ser Gly Leu Phe Thr Val Phe Thr Gly 210 215 220 Val Lys Asn Phe Ser Arg lie His Leu Val Asp Lys Trp Asn Gly Val 225 230 235 240 Ser Lys Val Asn Tyr Trp His Ser Asp Gin Cys Asn Met lie Asn Gly 245 250 ■ 255 Thr Ser Gly Gin Met Trp Ala Pro Phe Met Thr Pro Glu Ser Ser Leu 260 265 270 Glu Phe Tyr Ser Pro Glu Ala Cys Arg Ser Met Lys Leu Val Tyr Lys 275 280 285 Glu Gin Gly Val Phe Gly Gly lie Pro Thr Phe Arg Phe Val Ala Pro 290 295 300 Ser Thr Leu Phe Ala Asn Gly Ser Val Tyr Pro Pro Asn Glu Gly Phe 305 310 315 320 Cys Pro Cys Arg Glu Ser Gly lie Gin Asn Val Ser Thr Cys Arg Phe 325 330 335 Asn Ala Pro Leu Phe Leu Ser His Pro His Phe Tyr Asn Ala Asp Pro 340 345 350 Val Leu Ala Glu Ala Val Ser Gly Leu His Pro Asn Pro Lys Glu His 355 360 365 Ser Leu Phe Leu Asp lie His Pro Val Thr Gly lie Pro Met Asn Cys 370 375 380 Ser Val Lys Leu Gin Leu Ser Leu Phe Val Lys Ser Val Lys Gly lie 385 390 395 400 Gly Gin Thr Gly Asn lie Gin Pro Val Val Leu Pro Leu Met Trp Phe 561999 89 405 410 415 Glu Glu Ser Gly Ala Met Glu Gly Glu Thr Leu Glu Thr Phe Tyr lie 420 425 430 Gin Leu Val Leu Met Pro Lys Val Leu His Tyr Ala Gin Tyr Val Leu 435 440 445 Leu Ala Leu Gly Cys Val Leu Leu Leu lie Pro lie lie Tyr Gin lie 450 455 460 Arg Ser Gin Glu Lys Cys Tyr Leu Phe Trp lie Ser Phe Lys Lys Gly 465 470 475 480 Ser Lys Asp Lys Glu Ala Val Gin Ala Tyr Ser Glu Phe Leu Met Thr 485 490 495 Ser Ala Pro Lys Gly Thr Val Leu Gin Glu Ala Arg Leu 500 505 <210> 5 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 5 gcacaggccc atgttttact <210> 6 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 6 gtgatagagg cagcgactcc <210> 7 <211> 10 <212> DNA <213> Artificial <223> Synthetic <400> 7 gcacaggccc <210> 8 <211> 27 <212> DNA <213> Artificial <223> Synthetic <400> 8 ctcctcaccg cttcattcta aactgtt <210> 9 <211> 22 <212> DNA <213> Artificial <223> Synthetic <4 00> 9 agtctcaagc actggagcac eg 561999 90 <210> 10 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 10 aactctggag cagcatgtgt caacagc 27 <210> 11 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 11 actccaggaa ggacaccgtg tcattgt 27 <210> 12 <211> 25 <212> DNA <213> Artificial <223> Synthetic <400> 12 ctggagaagg aaatggcaac ctaat 25 <210> 13 <211> 25 <212> DNA <213> Artificial <223> Synthetic <4 00> 13 aacttctcac tgggccacac ttacc 25 <21C> 14 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 14 agggatcctt ctaggcctga ccacgtc 27 <210> 15 <211> 27 <212> DNA <213> Artificial <223> Synthetic <400> 15 gctacacagg agatcctcgt tggttgt 27 <210> 16 <211> 23 <212> DNA <213> Artificial <223> Synthetic <400> 16 tgactcccca tgtcctttac ctc 23 <210> 17 <211> 26 <212> DNA <213> Artificial 561999 91 <223> Synthetic <4 00> 17 gccacttggt tcagctctga gcatcc 26 <210> 18 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 18 gggcagtgtc caagtcttta tgagttc 27 <210> 19 <211> 23 <212> DNA <213> Artificial <223> Synthetic <400> 19 cagcaacaga tacacacggc aca 23 <210> 20 <211> 27 <212> DNA <213> Artificial <223> Synthetic <400> 20 gtggtggagg cagggagaca cataagg 27 <210> 21 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 21 cctggcaggg agaggagaaa gttgagt 27 <210> 22 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 22 gcttggtaaa taatcacaca ccgaatg 27 <210> 23 <211> 27 <212> DNA <213> Artificial <223> Synthetic <400> 23 aaatcccaca tccctcccca gacttga 27 <210> 24 <211> 24 <212> DNA <213> Artificial <223> Synthetic <400> 24 gaccagggag agggggatta gaag 2 4 <210> <211> 27 <212> DNA <213> Artificial <22 3> Synthetic <4 0 0> tgaggcatca gacacatgac cacaagt <210> 26 <211> 26 <212> DNA <213> Artificial <223> Synthetic <4 00> 26 ccacattgtt tgttagctgg gtacat <210> 27 <211> 23 <212> DNA <213> Artificial <223> Synthetic <400> 27 cactgttgta cgcgctgtac ctg <210> 28 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 28 tgttgtgtgg ccctggagga catagag <210> 29 <211> 27 <212> DNA <213> Artificial <223> Synthetic <4 00> 29 aagcctttaa gggtgccaga gttctae <210> 30 <211> 22 <212> DNA <213> Artificial <223> Synthetic <4 00> 30 tcttgggtga agggcactat ca <210> 31 <211> 23 <212> DNA <213> Artificial <223> Synthetic <400> 31 gccacagtcc ataggatcgc aca <210> 32 <211> 27 <212> DNA <213> Artificial 561999 92 27 26 23 27 27 22 <223> Synthetic <400> 32 tcttttctca tctcaggctt gattggt <210> 33 <211> 21 <2l2> DNA <213> Artificial <223> Synthetic <4 00> 33 gtcaacccag gagccaccga t <210> 34 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 34 cccataaaca ggctgttaca <210> 35 <211> 19 <212> DNA <213> Artificial <223> Synthetic <400> 35 catctcaggc ttgattggt <210> 35 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 36 gggaatttat tacgacagca <210> 37 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 37 gcagggagac acataaggag <210> 38 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 38 aacgccctcc agcctcggac <210> 39 <211> 19 <212> DNA <213> Artificial <223> Synthetic <400> 39 cgccccttcg cccatgctc 561999 93 27 21 19 561999 94 <210> 40 <211> 18 <212> DNA <213> Artificial <223> Synthetic <4 00> 40 ccttgcccat agtcagag 18 <210> 41 <211> 20 <212> DNA <213> Artificial <223> Synthetic <400> 41 ccttttggag ccttgtggta 20 <210> 42 <211> 18 <212> DNA <213> Artificial <223> Synthetic <400> 42 tggtgccctc aatcatca 18 <210> 43 <211> 22 <212> DNA <213> Artificial <223> Synthetic <400> 43 catgttgaaa gacaggctgt tg 22 <210> 44 <211> 74 <212> DNA <213> Artificial <223> Synthetic <4 00> 44 tggtgccctc aatcatcaag cagcaggtcc tcaagaatgt gcgcatcgac cccaacagcc 60 tgtctttcaa catg 7 4 561999 95

Claims (28)

1.CLAIMS 1. A method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or (3-carotene content, which comprises the step of determining the scavenger receptor class 1, member 1 (SCARBl) allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the SCARBl allelic profile.

2. A method as claimed in claim 1, wherein the milk p-carotene content is milk fat p-carotene content.

3. A method as claimed in claim 1, wherein the allelic profile is determined with respect to DNA, mRNA and/or protein obtained from said bovine.

4. A method as claimed in claim 1, wherein the allelic profile is determined by determining the presence or absence of the C allele at the C-321 G promoter polymorphism of the bovine SCARB1 gene.

5. A method as claimed in claim 1, wherein the allelic profile is determined by determining the presence or absence of the G allele at the C-321 G promoter polymorphism of the bovine SCARBl gene.

6. A method as claimed in claim 4 or claim 5 wherein the allelic profile of the C-321G promoter polymorphism in the SCARBl gene is determined by the use of one or more polymorphisms in linkage disequilibrium with this polymorphism.

7. A method as claimed in claim 1 wherein the allelic profile is determined by determining the expression or activity of a SCARBl gene or gene product.

8. A method of identifying or selecting a bovine having a desired SCARBl allelic profile comprising determining the allelic profile according to the method of any one of claims 1 to 7 and identifying or selecting said bovine on the basis of the determination.

9. A method of determining genetic merit of a bovine with respect to milk or tissue colour or p-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or p-carotene content, the method comprising providing data about the SCARBl allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the data. 561999 96

10. A method for identifying or selecting a bovine with respect to milk or tissue colour or p-carotene content, or with respect to capability of producing progeny that will have one or more desired milk or tissue colour or p-carotene content traits, the method comprising providing data about the SCARBl allelic profile of said bovine, and identifying or selecting the bovine on the basis of the data, wherein the the SCARBl allelic profile comprises data indicative of the presence or absence of one or more alleles at the C-321G promoter polymorphism in the SCARBl gene, or one or more polymorphisms which are in linkage disequilibrium with the C-321G promoter polymorphism in the SCARBl gene.

11. A method as claimed in claim 9 or 10, wherein the milk or tissue colour or [3-carotene content is increased milk colour or increased milk p-carotene content.

12. A method as claimed in any one of claims 9 to 10, wherein the milk or tissue colour or (i-carotene content is increased tissue colour or increased tissue (3-carotene content.

13. A method as claimed in claim 11 or 12 comprising determining (a) the presence of the C allele at the C-321G promoter polymorphism in the SCARBl gene, or (b) the absence of the G allele at the C-321G promoter polymorphism in the SCARBl gene, or (c) both (a) and (b), and identifying or selecting the bovine on the basis of the determination.

14. A method as claimed in claim 9 or 10, wherein the milk or tissue colour or (3-carotene content is decreased milk colour or decreased milk p-carotene content.

15. A method as claimed in claim 9 or 10, wherein the milk or tissue colour or p-carotene content is decreased tissue colour or decreased tissue P-carotene content.

16. A method as claimed in claim 14 or 15 comprising determining (a) the absence of the C allele at the C-321G promoter polymorphism in the SCARBl gene, or (b) the presence of the G allele at the C-321G promoter polymorphism in the SCARBl gene, or (c) both (a) and (b), 561999 97 and identifying or selecting the bovine on the basis of the determination.

17. A method as claimed in claim 10, comprising the step of amplifying at least a fragment of the bovine SCARBl gene sequence to determine the presence or absence of the one or more alleles.

18. A method as claimed in claim 17, wherein the primers used in the amplification are selected from the group consisting of SEQ ID NOs: 5 to 44.

19. A method as claimed in claim 17 or 18 wherein the presence or absence of the one or more alleles is determined by determining the expression or activity of a SCARBl gene or gene product.

20. A probe or primer having at about at least 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 3, and comprising a cytosine at the position corresponding to the C-321G promoter polymorphism in the SCARBl gene, or comprising a nucleotide capable of hybridising to a nucleotide capable of hybridising under stringent conditions to a cytosine at the position corresponding to the C-321G promoter polymorphism in the SCARB1 gene.

21. A probe or primer having about at least 12 contiguous bases of one of SEQ ID NOs: 5-44.

22. A probe or primer comprising a nucleotide sequence having at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 3 wherein the about 12 contiguous bases comprise or are within about 1 to about 2000 nucleotides of the C-321G promoter polymorphism in the SCARBl gene.

23. A pair of primers comprising two primers as claimed in claim 22.

24. Collected semen produced by a bovine identified by the method of any one of claims 8 or 10 to 19.

25. A method of selecting a herd of bovine, comprising selecting individuals by the method of any one of claims 8 or 10 to 19, and segregating and collecting the selected individuals to form the herd.

26. A herd of bovine selected by the method of claim 25.

27. A herd of bovine comprising two or more bovine, wherein the bovine are the progeny of one or more bovine selected by the method of any one of claims 8 or 10 to 19.

28. Collected or pooled milk produced by bovine identified by the method of any one of claims 8 or 10 to 19. 561999 98 35. Collected or pooled milk produced by bovine as claimed in claim 26. Collected or pooled milk as claimed in claim 28 or 29 having increased or decreased milk colour or increased or decreased (3-carotene content when compared to milk produced by a bovine having a SCARBl gene comprising the nucleotide sequence of SEQ ID NO: 1. A dairy product made from the milk as claimed in any one of claims 28 to 30. A kit for genotyping a bovine with respect to one or more milk or tissue colour or p-carotene content traits, comprising a probe or primer as defined in any one of claims 21 to 23 or a pair of primers as defined in claim 23. An isolated, purified or recombinant nucleic acid molecule comprising nucleotide sequence selected from (a) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising the C-3 21G promoter polymorphism; or (b) any one or more of SEQ ID NOs:5 - 44; or (c) a complement of (a) or (b); or (d) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (c) under stringent conditions. A vector comprising the nucleic acid of claim 33. A method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or p-carotene content, the method comprising determining milk or tissue colour or P-carotene content of the bovine, determining the SCARBl allelic profile of the bovine, comparing the SCARBl allelic profile of the bovine or the milk or tissue colour or p-carotene content of the bovine with that of a bovine having a known SCARBl allelic profile; determining the genetic merit of the bovine on the basis of the comparison. AQiMTI FOR TH1 APPySAKf 561999 99 ABSTRACT The present invention provides methods of genotyping bovine for desired milk or tissue colour or p-carotene content traits by determining the SCARBl genotype of said bovine, and particularly wherein the presence or absence of the C allele or of the G allele at the C-321 G promoter polymorphism in the SCARBl gene is associated with variation in milk or tissue colour or P-carotene content.