DETERMINATION OF GENOMIC SEX IN SALMONIDS BACKGROUND OF THE INVENTION

The present invention relates to the isolation of a genetic marker useful for determining the sex of fish belonging to the family salmonidae (sal onids) . In particular it relates to the isolation of a growth hormone pseudogene (GH-S_) , which is linked to a sex-determining locus on the Y chromosome and provides a marker useful for determining the genomic sex of fish.

Because of the depletion of natural stocks of salmon, aquaculture represents an increasingly dominant component of the commercial salmon fishery. For example, aquaculture accounts for the total commercial production of atlantic salmon (Salmo salar) on the Atlantic Coast of Canada, while on the Pacific coast, the production of chinook salmon (Oncorhynchus tshawytscha) through aquaculture exceeds the combined commercial and recreational catch of wild and hatchery produced chinook salmon

The rapid growth of chinook salmon aquaculture is closely linked to the development and implementation of sex control biotechnologies for this species (Solar, et al . Aguaculture ' 89, World Aqua . Soc , Los Angeles, CA. 12-16

Feb . (1989). Female chinook salmon mature at 3, 4, or 5 years of age, while males mature on average one year earlier at 2, 3, or 4 years. The culture of monosex female populations thus provides the aquaculturist with a greater window within which to market this species prior to the development of secondary sexual characteristics. In. addition, the use of monoculture female populations avoids the precocious sexual maturation of male fish as grilse which results in significant losses to the farmer due to an unmarketable or low-value product. For these reasons, virtually all of the chinook salmon grown in British Columbia are monosex females.

Monosex populations have been produced by sex- reversing normal (XX) female salmonids by androgen treatment during early development to generate phenotypic males that produce only X-bearing sperm. Hunter, et al . , Aquaculture , 33: 355-364 (1983) . This X-bearing sperm is used to fertilize normal ova resulting in all-female offspring for use in production facilities. A small number of XX zygotes can be masculinized to maintain a broodstock of monosex males.

This monosex sperm technology can be combined with triploidy induction to produce monosex female triploid salmonids which are reproductively sterile. It is desirable to maintain sterile salmon because of the increasing concern regarding the potential for genetic interaction between escaped farm fish and wild fish. This is of particular concern where the farmed fish are genetically altered through selection or transgenesis.

The implementation and maintenance of monosex populations requires that genetic and phenotypic sex be independently discernible such that Y-chromosome-bearing males can be distinguished from phenotypic males which carry the female genotype (XX) . Traditional methods of sexual determination include identification of morphological differences, or, where there is little sexual dimorphism, examination of gonad tissue. These means however, inherently rely on phenotypic characteristics.

Recently, a marker (designated OtYl) has been identified that indicates the genomic sex of chinook salmon independently of the phenotypic sex (Devlin, et al . Can. J. Fish . Aquat . Sci . , 48: 1606-1612 (1991). This marker however, appears to be restricted to chinook salmon. Moreover, OtYl is a repetitive DNA which occurs in several hundred copies. This may tend to confuse results in hybridization and PCR experiments. In addition, since most organisms carry many different types of repetitive DNA in their genome, probes for OtYl may tend to hybridize with DNA from other species.

The present invention provides a novel marker useful for determining the sex of a number of sal onid species which overcomes these limitations. Moreover, the marker possesses

unique features that facilitate the rapid detection of its presence or absence in a particular fish.

SUMMARY OF THE INVENTION The present invention provides an improved method of determining the geno ic sex of a salmonid by detecting the presence or absence of the growth hormone pseudogene GH-_.. The pseudogene is detected by amplification of a select subsequence specific to the pseudogene, or by duplex formation of a nucleic acid which hybridizes specifically to the pseudogene and to no other gene in the genome of the salmonid species. The method is useful in a number of salmonids, in particular Oncorhynchus tεhawytscha and Oncorhynchus klsutch . Where the method involves DNA amplification, such amplification may be accomplished by polymerase chain reaction or ligase chain reaction.

The method may involve identifying the presence or absence of a deletion of about 149 base pairs between exons 5 and 6 in the growth hormone pseudogene of salmonids. One aspect of the invention relates to pairs of PCR primers for use in detecting the presence or absence of the GH- . The PCR primers are competent to amplify a DNA sequence which includes a subsequence of exon 5 and intron 5 of the growth hormone gene and pseudogene, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 4, intron 4, or exon 5 and the other primer of said pair binds selectively to conserved regions of exon 6, said conserved regions present in both the growth hormone gene and the growth hormone pseudogene. The invention also provides for a pair of PCR primers competent to amplify the DNA sequence between about base 4870 and base 5019 of Sequence Id No: l, designated GH-I, wherein one primer of said pair of PCR primers binds selectively to conserved regions of exon 5, and the other primer of said pair binds selectively to conserved regions of intron 5 or exon 6. Preferably the primer pair consists of an oligonucleotide of sequence 5'-CCTGGATGACAATGACTCTCA- 3• and an oligonucleotide of sequence 5'-CTACAGAGTGCAGTTGGCCTC- 3• .

Another aspect of the invention relates to probes capable of detecting the presence of the GH-Ψ pseudogene. The probes hybridize specifically to the GH-Ψ pseudogene, but not to the GH-I or GH-II genes in 2x SSC, 0.1% SDS at 42°C. The probes may be selected to hybridize specifically to the region between about base 4843 and base 4863 of Sequence Id No: 2 in 2x SSC, 0.1% SDS at 42°C. The probes may be labeled with a marker such as a fluorophore, a lumiphore, a chromogen, a radioactive label, horseradish peroxidase, biotin, or dioxigenin.

An additional aspect of the invention relates to kits useful for determining the sex of salmonids by detecting the presence or absence of the GH-Ψ pseudogene. These kits take a variety of forms and can comprise one or more containers containing a nucleic acid competent to specifically detect the GH-Ψ pseudogene. The nucleic acid may be a pair of PCR primers, as described above, competent to amplify a DNA subsequence which includes a portion of exon 5 and intron 5 of the GH gene or pseudogene. The nucleic acid may alternatively be a probe, as described above, which hybridizes to the GH-Ψ pseudogene, but not the GH-I or GH-II genes under stringent conditions.

Another aspect of this invention is an isolated nucleic acid which selectively binds to the Sequence Id No.: 2, designated a GH-Ψ pseudogene, said selective binding to occur in genomic libraries of salmon in O.lx SSC, 0.1% SDS at 42 ^β C. Additionally the invention includes a nucleic acid wherein said nucleic acid is Sequence Id No. : 2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows restriction maps of csGH-15, csGH-16 and csGH-2 EMBL clones containing GH genes. Solid boxes in phage DNA denote regions hybridizing to sGH-4. Exons are represented by black boxes. A TATA box, the translation initiation and termination codons, and the polyadenylation signal are indicated by TATA, ATG and TAG/TAA, and AATAAA respectively. Restriction sites are E, Eco RI; H, Hind III; S, Sal I; Sp, Sph I; (S) , Sal ^' I site derived from vector arms.

It is clear that the restriction map of csGH-15 differs from that of csGH-16. For example, the 6 kb Hind III fragment in csGH-15 covers the entire region which hybridized with the GH-I cDNA; while in csGH-16, there were three Hind III sites in the 6 kb positive region. This indicates that these two clones represented two different GH genes. The restriction map of csGH-2 differed from that of csGH-15 and csGH-16. However, comparing it with the map of csGH16 revealed a 7kb overlapped region, which suggested that the DNA sequences in these two clones were linked in the chromosome. Thus, a longer map spanning approximately 27.5 kb at the GH locus could be obtained by aligning the map of csGH-2 with that of csGH-16.

Figure 2 illustrates the detection of the GH-Ψ pseudogene and the GH-II gene in chinook salmon by PCR. Figure 2(A) illustrates the PCR strategy. Primers F and G are derived from part of the conserved sequences in exon V and VI of GH-I and GH-Ψ genes respectively. The distance between primers F and G in GH-I and GH-Ψ genes is 782 bp and 271 bp respectively. Figure 2(B) shows the analysis of the PCR products by agarose gel electrophoresis. The gel was stained with ethidium bromide. The three PCR fragments are designated as I, II and III. C, chinook salmon genomic DNA from a male fish; 16, plasmid clone of GH-I gene; 15, plasmid clone of GH-Ψ pseudogene; M, molecular weight markers (fX-174-RF DNA Hae III digest) .

Figure 3 illustrates the male-specific distribution of GH-Ψ gene in chinook salmon and coho salmon. Figure 3(A) shows the results of a PCR analysis, using primers F and G, of 50 chinook salmon (25 males and 25 females) . GH-Ψ is found present only in the males. Data from 11 males and 10 females are shown here. Figure 3(B) shows a chinook salmon family ^" analysis of the distribution of GH-Ψ by F/G PCR. One chinook salmon family containing both parents and five each of male and female offspring were used. Three GH genes (GH-I, GH-II and GH-Ψ) were found in males, two GH genes (GH-I and GH-II) in females. Figure 3(C) shows a family analysis of the distribution of the GH-Ψ in coho salmon (Oncorhynhus kiεutch) by PCR using the same set of primers.

Figure 4 shows the results of a genomic Southern blot to determine the number of GH genes present in chinook salmon. Genomic DNA from male chinook salmon was digested with Eco RI (Ev) , Sst I (Ss) and Sea I (Sc) , and probed with chinook salmon GH-I cDNA. The size of the positive bands was estimated by comparing to the molecular weight marker (lDNA-Hind III digest).

Definitions The phrase "specifically detect" as used herein refers to the process of determining that a particular subsequence is present in a DNA sample. A DNA sequence may be specifically detected through a number of means known to those of skill in the art. These would include, but are not limited to amplification of the particular target sequence through polymerase chain reaction or ligase chain reaction, hybridization of the sequence to a labeled probe, and binding by labelled ligands or monoclonal antibodies. For a discussion of various means of detection of specific nucleic acid sequences see Perbal, B. A Practical Guide to Molecular CLoning, 2nd Ed. John Wiley & Sons, N.Y. (1988) which is incorporated herein by reference.

The phrase "select subsequence" is used herein to refer to a particular DNA subsequence that is of interest. It is often a predetermined or known sequence of nucleic acid bases. A select subsequence is typically chosen because of a unique sequence identity. Typically a select subsequence is one targeted for DNA amplification and often is useful as a specific marker for the presence of a particular gene. The term "genomic sex" refers to the sex of the fish as indicated by its genotype rather than its phenotype. A male salmonid typically has one X and one Y chromosome (XY) , while a female salmonid contains two X chromosomes (XX) . Because female salmonids may be masculinized to produce sperm that are genotypically female (XX) phenotypic sexual characteristics may not be diagnostic of genotypic sex.

The term "salmonid" refers to species which are members of the teleost fish family salmonidae. Salmonids

include, but are not limited to Atlantic salmon (Salmo salar) , Chinook Salmon (Oncorhynchus tschawytscha ) , Coho salmon (Oncorhynchus kisutch ) , Chum salmon (Oncorhynchus keta) , Pink salmon (Oncorhynchus gorbuscha ) , Sockeye salmon (Oncorhynchus nerka) , and Rainbow trout (Oncorhynchus ykiss) .

The term pseudogene is used herein to refer to a growth hormone (GH) gene which cannot be correctly spliced and translated to yield a functional GH. Features of pseudogenes which prevent correct splicing and translation include, but are not limited to, premature stop codons, deletions, and incorrect splicing signals at intron/exon junctions.

The term "subsequence" refers to a sequence of a nucleic acid that comprises a part of a longer sequence of a nucleic acid. The phrase "conserved region(s)" is used herein to refer to subsequences of the GH genes which differ by less than about 5% of their base composition between the various salmonid GH genes when optimally aligned using conventional sequence analysis computer programs such as CLUSTAL (PC/Gene) , GAP or BESTFIT, etc.

The term "oligonucleotide" refers to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides. Oligonucleotides may include, but are not limited to, primers, probes, nucleic acid fragments to be detected, and nucleic acid controls. The exact size of an oligonucleotide depends on many factors and the ultimate function or use of the oligonucleotide. Oligonucleotides can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth. Enzvmol. 68.:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzvmol. 6_:109-151; the diethylphosphoramidite method of Beaucage et al., 1981, Tetrahedron Lett. 22:1859-1862; and the solid support method of U.S. Patent No. 4,458,066.

The term "primer" refers to an oligonucleotide, whether natural or synthetic, capable of acting as a point of initiation of DNA synthesis under conditions in which

synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. A primer is preferably a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 25 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template.

The phrase "PCR primers competent to amplify" as used herein refers to a pair of PCR primers whose sequences are complementary to DNA subsequences immediately flanking the DNA subsequence (target sequence) which it is desired to amplify. The primers are chosen to specifically bind those particular flanking subsequences and no other sequences present in the sample. The PCR primers are thus chosen to amplify the unique target sequence and no other.

The term "probe", as used herein, refers to a molecule which binds to a specific (select) subsequence of a nucleic acid. A probe is preferably an oligonucleotide which binds through complementary base pairing to a subsequence of a target nucleic acid. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labelled as with isotopes, chromophores, lu iphores, chromogens, or indirectly labelled such as wrth biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select subsequence. The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can

function in a similar manner as naturally occurring nucleotides.

The terms "hybridize" or "hybridizing" refer to the binding of two single stranded nucleic acids via complementary base pairing.

The phrase "binding selectively" refers to complementary hybridization between an oligonucleotide and a target sequence and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired priming for the PCR polymerases.

"Amplifying" or "amplification", which typically refer to an "exponential" increase in target nucleic acid, are being used herein to describe both linear and exponential increases in the numbers of a select target sequence of nucleic acid.

The phrase "hybridizing specifically to", refers to the binding of a molecule only to a particular DNA sequence under stringent conditions.

The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a complementary probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.2 molar at pH 7 and the temperature is at least about 60 ^β C Tor long sequences (e.g. greater than about 50 nucleotides) and at least about 42 ^β C for shorter sequences (e.g. 10 to 50 nucleotides) . The term "SSC" refers to a citrate-saline solution of 0.15 M sodium chloride and 20 mM sodium citrate. Solutions are often expressed as multiples or fractions of this concentration. For example, 6x SSC refers to a solution

having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate. Similarly, 0.2x SSC refers to a solution 0.2 times the SSC concentration or 0.03 M sodium chloride and 4 mM sodium citrate.

DETAILED DESCRIPTION The present invention provides a method for determining the genomic sex of various salmonids (family salmonidae) . In particular, the invention provides the nucleic acid sequence of a pseudogene which is linked to a sex determining locus on the Y chromosome, and may be used as a marker for determination of the sex of the fish.

Method of Detecting the GH-Ψ Pseudoσene

The method of the present invention relies on the discovery of a growth hormone gene, designated the GH-Ψ pseudogene which is linked to the sex-determining locus on the Y chromosome of various salmonids. An assay for the presence or absence of this pseudogene, in effect, assays for the presence or absence of the Y chromosome and therefore indicates the genetic sex of the fish.

The assay generally comprises isolating a DNA sample from a particular fish and then contacting that DNA with a nucleic acid competent to specifically detect the GH-Ψ. Two strategies may be utilized for detecting the presence or absence of the GH-Ψ pseudogene. The first strategy involves detection of duplexes formed by a probe which hybridizes specifically to the pseudogene and to no other DNA in the sample. The second strategy involves amplification of GH genes or subsequences and detection of the presence or absence of GH-Ψ by the presence or absence of particular length subsequences among the amplified products.

Isolation of DNA for Detection of GH-Ψ

In a preferred embodiment, DNA will be obtained from species which are members of the family salmonidae. Salmonids include, but are not limited to Atlantic salmon (Salmo salar) ,

Chinook Salmon (Oncorhynchus tschawytscha ) , Coho salmon (Oncorhynchus kisutch) , Chum salmon (Oncorhynchus keta) , Pink salmon (Oncorhynchus gorbuscha) , Sockeye salmon (Oncorhynchus nerka) , and Rainbow trout (Oncorhynchus mykiss) . The method of the present invention is particularly of use in determining the sex of Coho and Chinook salmon. Of course, one of skill would recognize that, given the sequence listings provided herein for GH-I, GH-II and GH-Ψ, it is a relatively straightforward to determine whether the GH-Ψ pseudogene exists in other fish species.

The sexing of a fish using a DNA marker requires that the DNA sequence be accessible to the particular probes used or to the components of the amplification system if the DNA sequence is to be amplified. In general, this accessibility is ensured by isolating the nucleic acids from the sample. DNA may be isolated from a variety of tissues including liver, muscle, blood, and sperm, fins and scales. The choice of tissue is dictated by a number of factors including the quantity of starting material required, the difficulty of isolating DNA from a particular tissue, the possible presence of particular contaminants in a given tissue, and whether or not it is desired that sampling be non- lethal. The choice of tissue is readily apparent to one of skill in the art. In fish, sperm, liver and blood are frequently used as sources of DNA because they provide relatively large amounts of material. In particular, blood provides a tissue which is easily sampled without harm to the organism and, from which DNA is easily extracted.

A variety of techniques for extracting nucleic acids from biological samples are known in the art. For example, see those described by Sambrook et al .. Molecular Cloning ^~ - A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1985), by Han, et al . Biochemistry, 26: 1617-1625 (1987) and by Du, et al . Bio /Technology, 10: 176-181 (1992) which are incorporated herein by reference.

Alternatively, if the sample is fairly readily disruptable, the nucleic acid need not be purified prior to amplification by the PCR technique, i.e., if the sample is

comprised of cells, particularly peripheral blood lymphocytes or monocytes, lysis and dispersion of the intracellular components may be accomplished merely by suspending the cells in hypotonic buffer or boiling them in a low concentration of alkali (i.e. 10 mM NaOH) .

Detection of GH-Ψ Using Hybridization Probes

In one embodiment the GH-Ψ pseudogene may be detected by contacting DNA obtained from the salmonid with a probe which specifically binds the entire GH-Ψ gene or a subsequence of that gene and does not specifically bind to any other DNA sequences in the sample. While such probes may be proteins, oligonucleotide probes are preferred. Typically, the sequence of the oligonucleotide probe is chosen to be complementary to a select subsequence; a subsequence unique to GH-Ψ whose presence or absence is to be detected. Under stringent conditions the probe will hybridize with the select subsequence forming a stable duplex.

The probe is typically labeled and detection of the label in association with the target DNA indicates the presence of the GH-Ψ pseudogene. The probe may be used to detect GH-Ψ directly in a DNA sample without amplification of the GH-Ψ subsequences. For example, unamplified DNA sequences may be probed using a Southern blot. The DNA of the sample is immobilized, on a solid substrate, typically a nitrocellulose filter or a nylon membrane. The substrate-bound DNA is then hybridized with the labeled probe under stringent conditions and unhybridized probe is then washed away. Labeled probe detected in association with the immobilized GH sequences (e.g. bound to the substrate) indicates the presence of the GH-Ψ gene. Means for detecting specific DNA sequences are- well known to those of skill in the art. Protocols for Southern blots as well as other detection methods are provided in Maniatis, et al . Molecular Cloning: A Laboratory Manual . Cold Spring Harbor Laboratory Press, NY (1982) which is incorporated herein by reference.

In another embodiment, the growth hormone gene subsequences are themselves labeled. They are then

hybridized, under stringent conditions, with a probe which is immobilized on a solid substrate. Detection of the label in association with the immobilized probe then indicates hybridization of the DNA and the presence of the GH-Ψ pseudogene.

In a preferred embodiment, the GH sequences or subsequences may be amplified by a variety of DNA amplification techniques (for example via polymerase chain reaction, ligase chain reaction, transcription amplification, etc.) prior to detection using a probe. Because the copy number of GH genes is low, the use of unamplified GH DNA results in an assay of low sensitivity. Amplification of GH DNA increases sensitivity of the assay by providing more copies of possible target subsequences. In addition, by using labeled primers in the amplification process, the GH DNA sequences may be labeled as they are amplified.

Selection of Probes for Detection of the GH-Ψ Pseudogene

Full length sequences are provided for the GH-I gene in Sequence Listing No: l, and for the GH-Ψ gene in sequence listing No: 2. In addition a partial sequence listing for GH- II is provided in Sequence listing No: 3. Using these sequence listings, one of skill in the art may easily determine appropriate probes or primers for the detection of the presence or absence of the GH-Ψ pseudogene.

The GH-Ψ pseudogene (Seq. Id. No.: 2) provides a unique marker linked to the sex-determining locus on the Y chromosome. GH-Ψ is closely related to the GH-I and GH-II growth hormone genes in salmon and is most easily described by comparison to the GH-I growth hormone gene.

The GH-I gene consists 6 exons and 5 introns spanning 6.1 kb, including 1.9 kb of 5' flanking sequence, 4.1 kb representing a complete GH transcriptional unit, and 64 bp of 3' flanking sequence. Sequences at the exon-intron junction are consistent with the consensus sequences (GT-AG) at exon-intron boundaries. The size of the 6 exons of GH-I are 74 bp, 140 bp, 117 bp, 156 bp, and 147 bp and 543 bp respectively. A "TATA" box is found in 5 ¹ flanking sequence

of the GH-I gene 21 bp upstream from the transcriptional initiation site.

The GH-Ψ pseudogene (Seq. Id. No.: 2) resembles the GH-I gene. It has a "TATA" box, and contains six regions corresponding to the six exons in the GH-I gene. However GH-Ψ differs from GH-I in three respects: First, there is an incorrect splicing signal at the intron l/exon 2 junction, the junction sequence is changed to AC instead of the AG consensus. Second, one premature termination codon is found in exon 5, well before the correct translation stop codon site in exon 6. Third, an approximately 150 bp deletion is identified at the last half of exon 5 and the first part of intron 5; and the occurrence of this deletion event is supported by the identification of two 8 bp direct repeat sequences (GAACCTGG) at the 5* and 3' ends of the deletion.

This deletion is comparable to the region in GH-I between base 4870 and base 5019 of Sequence ID No.: 1. In addition, intron 5 of the GH-Ψ pseudogene is about 449 nucleotides shorter than intron 5 of the GH-I gene. A partial listing of the GH-II gene is provided in

Seq. Id No.: 3. GH-II is clearly distinct from the GH-I gene. However, it is almost identical to the corresponding sequence of chum salmon GH-II cDNA (Sekine, et al . , Blochim. Biophys . Acta . 1009: 117-120 (1989), with a characteristic change from Glnl35 in GH-I to Hisl35 in GH-II. In addition, there are eight silent mutations within this region compared with GH-I gene, and seven of them match with that in chum salmon GH-II gene. Id. These results confirm that fragment II was indeed amplified from the second GH gene, GH-II, in chinook salmon. One of skill would recognize that probes that specifically hybridize to the GH-Ψ pseudogene, but not to other GH genes under strict conditions may be readily constructed by exploiting the 150 bp deletion in GH-Ψ or other differences in sequence between the GH-Ψ pseudogene and the GH genes such as the incorrect splicing signal, or the premature stop codon. The design of hybridization probes is well known in the art. See, for example, Sambrook et al . , Molecular Cloning - A Laboratory Manual , Cold Spring Harbor

Laboratory, Cold Spring Harbor, New York, (1985) which is incorporated herein by reference.

In a preferred embodiment, the probe is an oligonucleotide sequence complementary to a subsequence spanning the deletion in GH-Ψ, the region around base 4853 in Sequence Listing No. : 2. In addition to complementarity to the subsequence spanning the deletion in GH-Ψ, the probe preferably has destabilizing mismatches with subsequences from other regions of the GH genes. In addition, the probe preferably spans the deletion symmetrically.

The exact length of the probe depends on many factors including the length of conserved regions in GH-I, GH-II, and GH-Ψ, the degree of sequence specificity desired, and the amount of internal complementarity within the probe. Such probes are preferably 17 to 25 bases in length. One of skill would recognize that longer probes would specifically hybridize at higher temperatures. For example, under stringent conditions, e.g. 2x SSC, 0.1% SDS at 42 ^β C, a 25 mer probe complementary to the subsequence spanning the region around base 3505 in Sequence Listing 2 would hybridize to the GH-Ψ pseudogene forming stable duplexes, but would not hybridize to any other nucleic acid sequence in the sample.

Oligonucleotide probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphotriester method of Narang et al., Meth . Enzymol . , 68:90-99 (1979); the phosphodiester method of Brown et al . , Meth. Enzymol . , 68:109- 151 (1979) ; the diethylphosphoramidite method of Beaucage et al . , Tetrahedron Lett . , 22:1859-1862 (1981); and the solid support method of U.S. Patent No. 4,458,066.

The probe may be labeled by attaching a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, the probe can be labeled at the 5'-end with ³² P by incubating the probe with ³ P-ATP and kinase (see Perbal, A Practical Guide to Molecular Cloning, 2nd ed. John Wiley, N.Y. (1988)). Other labels may be joined to the probe directly or through linkers. They may

be located at the ends of the probe or internally. Nonradioactive labels for a probe include fluorophores (e.g. fluorescein, rhodamine) , lu iphores (e.g luciferase) , chromogens (e.g red leuco dye) , and horseradish peroxidase (HRP) . Methods for labeling and detecting labeled probes are well known in the art and may be found, for example in Connell, et al . , Bio /Techniques 5: 342 (1987) which describes the attachment of fluorophores to oligonucleotides through an amide linkage, and U.S. Patent Nos. 4,914,210 and 4,962,029 which describe the use of HRP and are incorporated herein by reference. Kits for labelling oligonucleotides are widely available. See, for example, Boehringer Mannheim Biochemicals (Indianapolis, IN) for "Genius" labeling kits based on dioxigenin technology and Clonetech (South San Francisco, CA) for a variety of direct and indirect oligonucleotide labeling reagents.

Detection of GH-Ψ Through Amplification of Unigue Subsequences The GH-Ψ pseudogene while very similar to the growth hormone genes, contains a 149 bp deletion not present in the GH genes. The existence of this deletion may be exploited to detect the presence or absence of GH-Ψ pseudogene without the use of a hybridization probe. In this approach, subsequences are amplified which span the deletion in GH-Ψ and comparable regions in GH-I and GH-II. The resulting mixture contains amplified subsequences whose lengths reflect the presence or absence of the deletion and thus indicate the presence of the GH-Ψ pseudogene.

Typically three subsequences will be amplified in male fish and two subsequences will be amplified in female fish. Since a GH-I and GH-II gene is present in both male., and females, the resulting mixture will include at least two subsequences. The GH-I and GH-II subsequences are distinguishable from each other because the GH-II subsequence between exon 5 and exon 6 is approximately 380 bp shorter than the corresponding GH-I subsequence. In male fish, a third subsequence will be amplified from the GH-Ψ pseudogene. This subsequence is distinguishable from the GH-I and GH-II

subsequences because of a shorter intron 5 and by the presence of a 149 bp deletion. Thus, the amplified subsequences may be separated by size allowing determination of the presence or absence of the GH-Ψ pseudogene. Size separation may be accomplished by a variety of means known to those of skill in the art. These methods include, but are not limited to electrophoresis, density gradient centrifugation, liquid chromatography, and capillary electrophoresis. In a preferred embodiment, the fragments are separated by agarose gel electrophoresis. The bands are then stained with a marker to visualize them such as ethidium bromide and the gel is visualized under ultraviolet light.

As described above, an agarose gel typically shows 3 bands if the fish is male while only two bands if the fish is female (see Figure 3). If the amplified subsequences completely span intron 5, the bands will differ in length by about 509 bases between the GH-I and GH-Ψ subsequences and by about 380 bases between the GH-I and GH-II subsequences reflecting the differences in size of GH-I and GH-II and the shorter intron 5 and deletion in GH-Ψ.

Selection of Primers for Amplification of GH Marker Sequences

Amplification of GH genes or subsequences may be accomplished by methods well known in the art, which include, but are not limited to polymerase chain reaction (PCR) (Innis, et al . , PCR Protocols . A guide to Methods and Application . Academic Press, Inc. San Diego, (1990) which is incorporated herein by reference) , ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4: 560 (1989), Landegren, et al .. Science , 241: 1077 (1988) and Barringer, et al . , Gene , 89: 117 (1990) each of which is incorporated herein by reference) , ^"~~~ transcription amplification (see Kwoh, et al . , Proc. Natl . Acad. Sci . (U.S.A. ) , 86: 1173 (1989) which is incorporated herein by reference) , and self-sustained sequence replication (see Guatelli, et al . , Proc. Nat . Acad. Sci . (U.S.A. ) , 87:

1874 (1990) which is incorporated herein by reference) , each of which provides sufficient amplification so that the target sequence can be detected by nucleic acid hybridization to a

probe or by electrophoretic separation. Alternatively, methods that amplify the hybridization probe to detectable levels can be used, such as Qj3-replicase amplification. See, for example, Kramer, et al . Nature, 339: 401 (1989), Lizardi, et al . Bio /Technology, 6: 1197 (1988), and Lomell, et al . ,

Clin . Chem . 35: 1826 (1989) which are incorporated herein by reference.

In a preferred embodiment, amplification is by polymerase chain reaction using a pair of primers which flank and thereby amplify a selected GH subsequence. Selection of primers is readily apparent to one of skill in the art using the sequence listings of the present invention. For example, the entire GH gene may be amplified using widely spaced primers such as 5*-GAAAATGTTCAATGACT-3 • which is derived from exon 2 of the GH-I gene and 5 ^» -CTCTATCACTCTGAGCT-3 • which is derived from exon 6 of the GH-I gene. Alternatively, the primers may be chosen to amplify only the subsequences which clearly reflect differences between the GH-Ψ pseudogene and other GH genes in the sample. For example, the pair of primers consisting of 5•-CCTGGATGACAATGACTCTCA-3' , which is derived from a conserved sequence in exon 5 and primer G, 5'-CTACAGAGTGCAGTTGGCCTC-3' , which is from a conserved sequence in exon 6 flank and amplify the region spanning a portion of exon 5 and all of intron 5. This region incorporates the deletion in GH-Ψ and therefore reflects the greatest differences between GH-Ψ and GH-I and GH-II.

In order to avoid false negatives due to failure to prime subsequences on one of the GH genes, the primers are chosen to specifically bind regions of the GH genes that are conserved between GH-I, GH-II, and GH-Ψ. Thus subsequences will be amplified from all GH genes present in the sample.

Identification of Conserved GH Sequences

Conserved regions for the purposes of this invention are GH gene subsequences that share at least about 95% sequence identity. "Percentage of sequence identity" is determined by comparing two optimally aligned sequences or subsequences over a comparison window or span, wherein the

portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl . Math . 2 : 482 (1981) which is incorporated herein by reference, by the homology alignment algorithm of Needleman and Wunsch J. Moϊ . Blol . 48: 443 (1970) which is incorporated herein by reference, by the search for similarity method of Pearson and Lipman, Proc. Natl . Acad.

Sci . (U.S.A. ) 85: 2444 (1988) which is incorporated herein by reference, by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program, Intelligenetics, Mountain View, Califonria, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG) , 575 Science Dr. , Madison, WI) , or by inspection. In particular, methods for aligning sequences using the CLUSTAL program are well described by Higgins and Sharp in Gene , 73: 237-244 (1988) and in CABJOS 5: 151-153 (1989) both of which are incorporated herein by reference.

Determination of the Presence of the GH-Ψ Marker in Other Salmonid Species

The GH-Ψ pseudogene has been identified in chinook, coho, chum and pink salmon. It further appears to be sex- linked in all of these species. It is therefore reasonable to infer that GH-Ψ may be present in salmonid species not yet screened. One of skill would recognize that the same techniques utilized to identify the presence or absence of GH-Ψ for the purpose of determining the sex of a fish may be utilized to determine if GH-Ψ is present in other species of salmonid. For example, fish species can be screened by extracting DNA from blood, and using primer F (5--CCTGGATGACAATGACTCTCA-3 ¹ ) , which is derived from a conserved sequence in exon 5 and primer G, (5•-CTACAGAGTGCAGTTGGCCTC-3•) which binds a conserved sequence in exon 6 to amplify a subsequences of the GH genes which, when amplified from GH-Ψ incorporate the 150 bp deletion. The amplified subsequences may be separated according to their size as described above. Sequences amplified from fish carrying the GH-Ψ marker will include a pair of subsequences differing in length by about 380 base pairs reflecting the differences length of the subsequences between exon 5 and exon 6 of the GH-I gene and the GH-Ψ pseudogene.

Of course one of skill would recognize other methods of assaying for the presence of GH-Ψ using the primers and probes of the present invention. For example, one might select primer pairs to amplify subsequences containing other regions unique to GH-Ψ pseudogenes. These might include, but are not limited to, the incorrect splicing signal at the intron l/exon 2 junction or the premature termination codon in exon 5.

Alternatively, one of skill may construct hybridization probes specific to the GH-Ψ pseudogene, but not to the GH-I or GH-II genes. These probes may be specific for the region spanning the 149 base pair deletion in GH-Ψ, or alternatively, they may be specific to other regions of the GH-Ψ pseudogene that are not conserved in the GH-I or GH-II

genes. Such regions may be readily identified by one of skill, using the sequence listings provided herein.

In addition to oligonucleotide primers and probes, it is also possible to use growth hormone gene cDNA probes to isolate GH genes in other species. The probes may be specific to GH-Ψ pseudogenes. Alternatively, the probes may hybridize to all homologous GH genes. In the latter case, the genes, once isolated, may be sequenced. Analysis of the sequence data will reveal the presence or absence of GH-Ψ pseudogenes. Techniques for the use of cDNA probes to isolate genes and subsequent sequencing are well known in the art (See Maniatis, et al . Molecular Cloning: A Laboratory Manual . Cold Spring Harbor Laboratory Press, NY (1982)).

Determining if the GH-Ψ Pseudogene is Sex-Linked

Once it is determined that the GH-Ψ pseudogene is present in a particular fish species, it is necessary to verify that GH-Ψ is linked to a sex-determining locus on the Y chromosome. To accomplish this a number of both males and females of the species are screened by the one of procedures described above for detecting the presence or absence of a GH-Ψ pseudogene. The sex of the fish must then be verified using another approach such as examination of gonads. If the GH-Ψ pseudogene is sex-linked, it should co-segregate with the males.

To verify that the apparent association of the GH-Ψ pseudogene with males is not simply due to the occurrence or non-occurrence of a particular polymorphism rather than actual sex-linkage, the distribution of the GH-Ψ pseudogene must be examined within a family of fish. If the males bear the GH-Ψ pseudogene, but the females do not, it will indicate both that the marker is present in the fish and that it segregates as a sex-linked marker.

Kits for Determining the Sex of Salmonids

The present invention also relates to kits, multicontainer units comprising useful components for practicing the present method. A useful kit can comprise a

container containing a pair of primers competent to specifically detect the GH-Ψ pseudogene and/or a probe specific to the GH-Ψ pseudogene. In some cases, the probe may be fixed to an appropriate support membrane. The probe or the primers may be labeled.

Other optional components of the kit include, for example, a polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin) , the appropriate buffers for PCR or hybridization reactions, and pre-cast gels for electrophoresis. In addition to the above components, the kit can also contain instructions for carrying out the present method.

The following examples are offered to illustrate, but not to limit the present invention.

EXAMPLE 1 Detection of the Presence or Absence of GH-Ψ

To test for the presence of GH-Ψ genes, chinook salmon genomic DNA was analyzed by PCR. To isolate DNA from sperm, 2 μl of sperm was suspended in 100 μl of lx PCR buffer (50 mM KC1, 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl ₂ , 0.1% triton X-100) , followed by the addition of 3 μl of proteinase K (10 mg/ml) . The sample was incubated for 2 hours at 55 ^β C, and then mixed with 100 μl of 10 mM NaOH. The mixture was boiled for 3 min and centrifuged for 3 min.

About 100 ng of DNA (3 μl of the supernatant) was amplified by PCR using two GH specific primers designated F and G. Primer F, 5 ^• -CCTGGATGACAATGACTCTCA-3• , was derived from a conserved sequence in exon 5 in both GH-I and GH-Ψ, while primer G, 5 ^» -CTACAGAGTGCAGTTGGCCTC-3• , was from a conserved sequence in exon 6 in both genes. A positive control was provided using, 40 ng each of plasmid DNA for the GH-I gene and the GH-Ψ gene. The PCR reaction and the analysis of the PCR products were carried out essentially as described by Du, et al . Bio /Technology , 10: 176-181 (1992), which is incorporated herein by reference. PCR cycling

parameters were 1 min at 9 °C, 1 min at 60°C, and 2 min at 72°C.

Primers F and G, shown in Figure 2(A), were derived from the sequences in exon 5 and 6 respectively, separated by intron 5. These primers were chosen to take advantage of the observation that the size of this intron differs between GH-I gene and GH-Ψ. Thus, PCR using primers F and G, should generate two fragments of 782 bp and 273 bp from GH-I and GH-Ψ respectively (Fig. 2(A)). However, PCR analysis showed that, in addition to the 782 bp (fragment I) and the 273 bp (fragment III) bands, a third band approximately at 400 bp (fragment II) was amplified (Fig. 2(B)).

In order to clarify the nature of fragment II, and to confirm that fragments I and III were indeed amplified from GH-I and GH-Ψ respectively, the three PCR fragments were gel purified, treated with T4 DNA polymerase to create blunt ends, cloned into pUC18 Smal I site by blunt end ligation, and sequenced. The sequence data show that fragment I was indeed derived from the GH-I gene, similarly, the sequence of fragment III was identical to the corresponding region of GH-Ψ (data not shown) . The sequence of fragment II is shown in Sequence Id No. : 3. Comparison with the sequences of chinook salmon GH-I (Ot) , chum salmon (Ok) GH-I and GH-II, clearly shows that it is distinct from that of GH-I. This suggests that it represents a different GH gene. However, it is almost identical to the corresponding sequence of chum salmon GH-II cDNA, as described by Sekine, et al . , Biochem . Biophys . Acta. , 1009: 117-120 (1989), with a characteristic change from Glnl35 in GH-I to Hisl35 in GH-II. This change is also true for GH-II in rainbow trout. See Agellon, et al . , Proc. Natl . Acad . Sci . (U.S.A. ) , 85: 5136-5140 (1988) and Rentier-Delrue, et al . , DNA, 8: 109-117 (1989). In addition, there are eight silent mutations within this region compared with GH-I gene, and seven of them match with that in chum salmon GH-II gene described by Sekine, et al . , Biochim . Biophys . Acta . , 1009: 117-120 (1989) . All these results confirm that fragment II

was indeed amplified from a second GH gene, GH-II, in chinook salmon.

EXAMPLE 2 Distribution and Sex-Linkage of GH-Ψ DNA

To examine the distribution of the three GH genes in chinook salmon, 50 unrelated chinook salmon (25 males and 25 females) were analyzed by PCR using the GH specific primers F (5•-CCTGGATGACAATGACTCTCA-3 •) and G (5--CTACAGAGTGCAGTTGGCCTC-3 •) described in Example l and shown in Figure 2(A) . DNA was isolated from blood cells from individual fish. The DNA isolation and PCR amplification were carried out essentially as described by Du, et al. Bio /Technology, 10: 176-181 (1992). The PCR data showed sex-linked distribution patterns

(Fig. 3). Three DNA fragments corresponding to GH-I, GH-II and GH-Ψ were amplified from males, however, only two of these fragments corresponding to GH-I and GH-II were generated in females (Fig. 3(A)). This suggested that GH-Ψ may be male specific.

To confirm this observation, a chinook salmon family containing both parents and five each of male and female offspring were analyzed by PCR using the same set of primers. It was found that the male parent and their male offspring had the extra GH-Ψ gene, while the female parent and the female offspring lacked GH-Ψ (Fig. 3(B)), further demonstrating that GH-Ψ is male specific.

To test whether the GH pseudogene exists in other salmonids, the genomic DNA of males and females from pink, coho, sockeye, chum, and Atlantic salmon, and from rainbow trout were analyzed by PCR using primers F and G. The results showed that the coho salmon is similar to chinook salmon in that the male has three bands while the female has two bands. Family studies in coho salmon confirmed that the pseudogene is male specific (Fig. 3(C)).

EXAMPLE 3 __ι_i-i-»τ of GH Genes in Chinook Salmon

To verify that there are only three GH genes in chinook salmon, a genomic Southern analysis was performed. Chinook salmon genomic sperm DNA (20 μg) was digested with restriction enzyme Eco RV, Sst I and Sea I separately. The digested DNA was separated by 0.7% agarose gel in lx TNE buffer at 65 mA for 6 hours. The DNA was transferred to Hybond-N nylon membrane (Amersham) using lOx SSC buffer for 24 hours. The filter was prehybridized with buffer containing 10% dextran sulfate, l M NaCl, 1% SDS, 50% forma ide and 200 mg/ml of sheared calf thymus DNA for 20 min at 42°C and followed by hybridizing with ³² P labeled complete chinook salmon GH cDNA in the same buffer system. The hybridization was carried out for 18 hours at 42°C. After hybridization, the filters were washed once with 2xSSC (0.3 M NaCl, 0.034 M sodium citrate) for 15 min at room temperature, twice with 2xSSC, 0.1% SDS for 15 min at 65°C, and once with O.lxSSC, 0.1% SDS for 10 min at 65°C. The filters were air dried at room temperature and exposed to X-ray film for 2 days at -70 ^β C.

As shown in Figure 4, four positive bands approximately at 5 kb, 7.5 kb, 18 kb and 27 kb were generated by digestion with Eco RV. Digestion with Sea I also gave four positive bands at approximately 5 kb, 8 kb, 20 kb, and 25 kb. The DNA sequence data shows that there is neither an Eco RV nor a Sea I recognition site in GH-I gene, so that only one fragment could be generated from GH-I gene by these two enzymes. However, there is one Eco RV and one Sea I recognition site in GH-Ψ. The Eco RV site is located in exon III, while the Sea I site is located in exon IV. Since Both of these sites are located in the middle of the gene, two positive fragments therefore could be generated from the pseudogene alone by a single digestion with either one of these two restriction enzymes. As a result, total of three fragments should be generated from GH-I and GH-Ψ. However, Southern analysis revealed four positive bands in both digestions, and the largest fragments by Eco RV or Sea I

digestion were 27 kb and 25 kb respectively. It is unlikely that either of these two 27 kb and 25 kb fragments contains more than one GH gene since restriction mapping showed that the GH-I gene locus spans at least 27.5 kb (Figure 1). Therefore, the extra positive band must come from a third GH gene, the GH-II gene. Overall, these results strongly suggest that there are only three GH genes in the male chinook salmon genome, i. e., GH-I, GH-II and a pseudogene, GH-Ψ.

EXAMPLE 4

Determination of the Sex of Oncorhynchus tshawvtscha DNA is isolated from the blood cells of an individual fish essentially as described by Du, et al . Bio /Technology, 10: 176-181 (1992) PCR amplification is carried out using oligonucleotide primers F and G which are 5•-CCTGGATGACAATGACTCTCA-3' and 5'-CTACAGAGTGCAGTTGGCCTC-3• respectively. The PCR reaction and the analysis of the PCR products is carried out essentially as described by Du, et al. (1992). PCR cycling parameters are l min at 94°C, 1 min at 60°C, and 2 min at 72°C.

The amplified PCR products are analyzed by agarose gel electrophoresis. The gel is stained with ethidium bromide to visualize the separated gene fragments.

The distance between primers F and G in GH-I and GH-Ψ and GH-II genes is 782 bp, 273 bp and about 400 bp respectively. Thus, amplified products from male fish produce three bands on an agarose gel, corresponding to subsequences from GH-I, GH-Ψ, and GH-II, while amplified products from female fish produce 2 bands corresponding to subsequences from GH-I and GH-II.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: A/F Protein

(B) STREET: 72 Bonard Road

(C) CITY: W. Newton

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) POSTAL CODE (ZIP): 02165

(G) TELEPHONE: (617) 357-9459 (H) TELEFAX: (617) 357-5548 (I) TELEX:

(ii) TITLE OF INVENTION: DETERMINATION OF GENOMIC SEX IN SALMONIDS

(iii) NUMBER OF SEQUENCES: 7

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(v) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO not yet assigned

(B) FILING DATE: 14-OCT-1994

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Weber, Kenneth A.

(B) REGISTRATION NUMBER: 31,677

(C) REFERENCE/DOCKET NUMBER: 16252-0002

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 543-9600

(B) TELEFAX: (415) 543-5043

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6136 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1904..1980

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1981..2383

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 2384..2523

( ix ) FEATURE :

(A) NAME/KEY: intron

(B) LOCATION: 2524..2659

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 2660..2776

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2777..3498

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3499..3654

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 3655..4786

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 4787.. 933

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 4934..5529

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 5530..5592

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(1971..1980, 2384..2523, 2660..2776, 3499

..3654, 4787..4933, 5530..5592)

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: join(2440..2523, 2660..2776, 3499..3654, 4787

..4933, 5530..5592)

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: join(1971..1980, 2384..2439)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..6136

(D) OTHER INFORMATION: /standard_name= "The Complete

Nucleotide Sequence of Chinook Salmon GH-I Gene"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 577..843

(D) OTHER INFORMATION: /note= "The 266 bp 5' flanking homologous region shared with the GH pseudo "

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1805..1808

(D) OTHER INFORMATION: /standard_name= "The CAAT Box"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1878..1885

(D) OTHER INFORMATION: /standard name= "The TATA Box"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 6051..6056

(D) OTHER INFORMATION: /standard_name= "The Polyadenylation Signal"

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

CCCCAAAACC AATTCTTTCT TTGGCCGCCT CTCCTTCCAG TTCTCTGCTG CCAATGACTG 60

GAACGAACTA CAAAAATCTC TGAAACTGGA AACACTTATC TCCCTCACTA GCTTTAAGCA 120

CCAACTGCCA GAGCAGCTCA CAGATTACTG CACCTGTACA TAGCCCACCT ATAATTTAGA 180

CCAAACAACT ACCTCTTTCC CTACTGTATT TAATTGATTT ATTTATTTTG CTCCTTTGCA 240

CCCCATTATT TTTATTTCTA CTTTGCACAT TCTTCCATTG CAAATCTACC ATTCCAGTGT 300

TTTACTTGCT ATATTGTATT TACTTTGCCA CCATGGCCTT TTTTTGCCTT TACCTCCCTT 360

ACTCTACCTC ATTTGCTCAC ATCGCATATA GACTTGTTTA TACTGTATTA TTGACTGTAT 420

GTTTGTTTTA CTCCATGTGT AACTCTGTGT CGTTGTATGT GTCGAACTGC TTTGCTTTAT 480

CTTGGCCAGG TCGCAATTGT AAATGAGAAC TTGTTCTCAA CTTGCCTACC TGGTTAAATA 540

AAGGTGAAAT ATTTTTTTTT AAATAAAATA AAATACAGTA TTTTTGATTT GCTGTACTCT 600

CAAAACAAGT CATCCCTACA TCCAATCTGA TAGTGTGGCC GAAACTAACC GTTGTATAGT 660

GCTCTTTTGA AAGCCAAGCT ACCACATATT CAACGAGGTA ATGAGGTCAG TAAATGCACT 720

TTCGGCCGCC CTTCCAAAGT GATGTGGGTT AACAGATCGA TTTCTGTTTT TGCCAGGAAA 780

GGATTAGGAC AGCGCTGGGC CTGACATTCA GCACGTCTCC GTGTCCTGAA CACTGAATAT 840

GGATCTAGGT TGAATATGTT CACCCAACTT AGGTTGAACA TGTTCACACC GGAACAGTCT 900

ACTCCCCCGT ATGCCACGCT GACATGATCT CATTACTGTC ATGACTATTG ACCAGATAAC 960

CTTTATAGGT GCCATGACAC CTTACACCTA CATGTGTATC GTAAACAATA CAGTAAGGTG 1020

GTGCGGCACA AACAGAGTGT AGGCTTTGGG CTGGTAACAT TGTGTAGTCA AGCACACATG 1080

CTGCTAGGCT GAAAGCAGTT CATGTGTTAT CCTCTGAGAT GTATACTTTT TTTAACAGCA 1140

TATACATTTT TCCCCCAAAA TCAATCAATC AATCAACGGA TGAAGTAGGG CCTGCACACT 1200

TGCTCCAATA GATACCGTTA TTAGGCTTTC TTGACAATGT TGAGATTACT GAAAGGATCT 1260

TCGTCAGGTA TGACAAAGGC TCTCACACAA AAAGAAAAAG GATCGGGTAT TTCACAGACT 1320

GGTATTGATC AAGTGACTCT TTATGTTGTG TGTGTTGATG ATAACAAAGA CCCTGTCTGA 1380

ATTTAAACAA AAAACTATAC ATTCTAACAT GTGCCGTCTC GAGTCCTTCT CTGTGTGTCT 1440

ACTTTGAGGA ATTTGACTAA GTGTTAATGC CATAGGACAT TCAATTTGAC ATTAAACAAT 1500

AACATATTGG GGTTAATAAA GAAGCAATAT AATAAATGTC TTGTCATACT GCCTGTTATC 1560

TACAGTACCA CAGCAGAATG GCAGAATAAC CTGTGTGTGT GTGTGTGTGT GTATCTTATG 1620

TCCATTCATT ACATCCTAGA CAACAGAGGT TTGTGTTGTA TGTGTTTTGA CCCTAATTCG 1680

TTCAGTCATC AAGTAAGTTG TTTTTTTAGG ACACCTCCCT CTTCCCAAAC TCATGGAAAA 1740

ATGTATGATT GATTTGACGT AATATGGTAA TTGTTCCGTC ATCACATACA AAAACAGGTC 1800

CTATCAATGA AAGGTGGTAA ATGGATGAAA ATGTCATGTT TCCTCCCATT GATACATTAA 1860

AACATGGATT CCCCATCTAT AAAAACAGTG GCCCCAAACA AACGACAACA TACTCAACCG 1920

ACCACCGCAC TTTCAAGTTA AGTAACCATC CTTGGCAATT AAGAGTAAAA ATG GGA 1976

Met Gly -22

CAA G GTAAGCCTGC TTTTTCTGTC TATTTCTTTT TTCAGTGGGA AGTCAGAGTA 2030

Gin

-20

CCATTTAGTA CAATTTAACT ACTGCTATGA GGTTATAATC TATTGACACA GAACCACCTG 2090

CTTTAACAAC CTAACTATGT GATCCATAAC ATTTACATTT TTGTCATTTA GCAGACACTC 2150

TTTTCCAGAG CGACTTACAT GAGCAATTGG GGTTACGTGC CTTGCTCAAG GGCACATCAG 2210

ATTTCTCACC TAGTCAGCTC TGGGGTTGAA ACCAGTAACC TTTCAGTTAC TGACCCAGCG 2270

CTCTTAACCA GCTAGGCTAT TGGTGTACGA TGGCTGGGAA AATCTTACTA AGGTATCTCA 2330

CCATAATTCG ACTTACTCGT TTTCTACATT TGTTATTTGA ATCTCTCTTT TAG TG 2385

Val

TTT CTG CTG ATG CCA GTC TTA CTG GTC AGT TGT TTC CTG AGT CAA GGG 2433 Phe Leu Leu Met Pro Val Leu Leu Val Ser Cys Phe Leu Ser Gin Gly -18 -15 -10 -5

GCA GCG ATA GAA AAC CAA CGG CTC TTC AAC ATC GCG GTC AGC CGG GTG 2481 Ala Ala lie Glu Asn Gin Arg Leu Phe Asn lie Ala Val Ser Arg Val 1 5 10

CAA CAT CTC CAC CTA TTG GCT CAG AAA ATG TTC AAT GAC TTT 2523

Gin His Leu His Leu Leu Ala Gin Lys Met Phe Asn Asp Phe 15 20 25

GTAAGACAGC TTTTGAATCT TCTTTGGACA TATCAAATAG TGTATCAATT ATTGTTCTTC 2583

TTCTTGTAGA CAGTGTCCTC TTCACACAAC CCTCGTGGCT AAAAGAATCT CTCTCTCCCT 2643

TTGTGATTTT GTGCAG GAC GGT ACC CTG TTG CCT GAT GAA CGC AGA CAG 2692

Asp Gly Thr Leu Leu Pro Asp Glu Arg Arg Gin 30 35

CTG AAC AAG ATA TTC CTG CTG GAC TTC TGT AAC TCT GAC TCC ATC GTG 2740 Leu Asn Lys lie Phe Leu Leu Asp Phe Cys Asn Ser Asp Ser lie Val 40 45 50 55

AGC CCA GTC GAC AAG CAC GAG ACT CAG AAG AGT TCA GTAAGTAACC 2786

Ser Pro Val Asp Lys His Glu Thr Gin Lys Ser Ser 60 65

TGGCTGAGAC AATTACGCAT GTTATGCCCT TTAGAACCAT ATAAAGTGTC AAATCGTGAC 2846

AGTTCCACTC TGCTATTCAC CTTAAATATG AACTCCTCCA TGATGCAAGA TTCCAAAAAT 2906

AAATAATAGG GCATCTCAAT TTGAACAATC GATAGAACTT AGTCATTAGT TATTGGGCAA 2966

GCAGACCACC AATTATGTAA ACTCAAATTT ATAATTTTTT ATTTAAATTT TATTTGAGCC 3026

TTTAATTAAC TTGGCAAGTC AGTTAAGAAC AAATTCTCAT TTACAATGAC AAGCAGAGGC 3086

AGCATCATGC ATGGCTCTCG AGTGGCACAG CAGTCTAAGG CACTACATCT CAGTGCCAGA 3146

GGTGTCACTG CAGACCCTAG TTCGATTCCA GACTGTATTT CAAACGGCTG TGATTGTGAG 3206

TCCATAGGGC GGCACACAAT TCTCCCAGCG TCGTTAGGGT TTGGCCGGGG TTGCAATACC 3266

TCAGTGTCTT CAACTAAGGT AGATAAAACA ACCACATATC ATTGCAAGTA AAACCATCAC 3326

TGTCTAATCG GTGGTTTCTC TATGTCTACA TTCTCTGTTT TGTGCTTTTC TGTACAGGAA 3386

ACCCACCCCA AAAGTATTTC ACTCAATCAT GTAAATAGGG CATCTCAAGC TGTAAATACA 3446

ACTCAACTTC ATTTTCCAAT AATCTGTGGT TTCTCTACAT CTTCACACAC AG GTC 3501

Val

CTG AAG CTG CTC CAT ATT TCT TTC CGT CTG ATT GAA TCC TGG GAG TAC 3549 Leu Lys Leu Leu His lie Ser Phe Arg Leu lie Glu Ser Trp Glu Tyr 70 75 80

CCT AGC CAG ACC CTG ATC ATC TCC AAC AGC CTA ATG GTC AGA AAC GCC 3597 Pro Ser Gin Thr Leu lie lie Ser Asn Ser Leu Met Val Arg Asn Ala 85 90 95 100

AAC CAG ATC TCT GAG AAG CTC AGC GAC CTC AAA GTG GGC ATC AAC CTG 3645 Asn Gin lie Ser Glu Lys Leu Ser Asp Leu Lys Val Gly lie Asn Leu 105 110 115

CTC ATC ACG GTAAATAATG GAGAGAGAAC AATGACCATT TGTGGTCTCA 3694 Leu lie Thr

CACTTTGTGC ACTGTAAACT CCAAGGCATT TTTAACTCAA ATACTTCTAG TAAGTTGAAC 3754

TCAAGGTCAA TGAAAAATCC TTATTGCTTA AAATGTTTAT GTGGTACTGG CTCAAAACTA 3814

AATGAGAAGT CACATCAATG CAATTTTTTA AAGTTATAAC AAATTCACTT TTACCAAGCA 3874

TGCTCTACTG CAGGTAGATT TAAAAAAAAA AAAAAAAAAA AAAAGAAGTT TTTAATGATC 3934

TGTGTTTTTG CATGTACAGA ACATTGAGTG ATTGATTCAT TTTATGCTAC ACAAAGATAT 3994

ATAACATACA TGTTTCAACG TTTTCATAAA GATGAACAAG TTACTAGAAT TTTGCAAACT 4054

CAACTTGCAG CCTGATGTGG CCTGTATACC GTGAGTTTCA GGCCACTGTA TTAGGGTAAA 4114

CTGACGCCTC AAAATAAGGT CTTATGAGAT AAGTAATGTA TTGTTGTAAA GAGTTGAATT 4174

CACTTGAAGG CCACAGGACT GAAAATGAAT GACAACAGCC ATGTCTCTGT CACTAACACA 4234

TACAGTCATG GGTGATAACT ACACTTTACT CAAAAAGGCC AGGCACACTG GGAAATTATA 4294

TTTGAGACGT GGCTTAGTGG GGGCATTACT AAAAAATGTC AAGCTGATAC AACTCAAATC 4354

TGGACTCATC ACAGGGTGAA TCTATAGGTT TGAGTAATTA CTGACTATAA TATCACTTTA 4414

AGTAACTGCA GTCAGATTCT GTATATTAAG TGCAACGGTT TCCTAAAAAG TGTTGAGTAA 4474

TGGCAGCACA TTGGGGTTTA CAGTGACATG AAAGGGAAAT ACCTGTATGC TTTCCTAGTT 4534

AGAAAGCATA GTGTAAGGAC CACGTATGCC TCTTCTCAGC AGATCTTTCA GGGCTTTACA 4594

TTGTGATGTG GTAACTGACC TTATCTATCA TCGTGATTAT ATCAGTGACA CCCCATTCAA 4654

TGACTGAATA TCGCCCCATT CAAGGACATT TATCCATGTG TCTTTTGCTA CGTGTGCTTT 4714

CAGAAAGGCC CAATAAACAA ATATTGATAT GCACACATCC ACCCCACCAT GCATCTCTCT 4774

CTGTCTCCAC AG GGG AGC CAG GAT GGC CTA CTG AGC CTG GAT GAC AAT 4822 Gly Ser Gin Asp Gly Leu Leu Ser Leu Asp Asp Asn 120 125 130

GAC TCT CAG CAA CTG CCC CCC TAC GGG AAC TAC TAC CAG AAC CTG GGG 4870 Asp Ser Gin Gin Leu Pro Pro Tyr Gly Asn Tyr Tyr Gin Asn Leu Gly 135 140 145

GGC GAC GGA AAC GTC AGG AGG AAC TAC GAG TTG TTG GCT TGC TTC AAG 4918 Gly Asp Gly Asn Val Arg Arg Asn Tyr Glu Leu Leu Ala Cys Phe Lys 150 155 160

AAG GAC ATG CAC AAG GTGCCGAACC ATGTTGCCTT CAATTTTATG TGCCTTCCTG 4973 Lys Asp Met His Lys 165

TATTTTCTAC AGTGCGTTGT TTTTTTGTAT TCTCTATTGC AAAGTATTGT TAGTAAATAA 5033

CTCACGGACA CTAGAGAAGC TTTAACCAAG TTTAATTCTT CCCAAAGGTT CTGTACAGCT 5093

ATAATCAGAC AGCAAAACTT TCTCACTCCA CAGTCATATA TATCCTACTT AAAACACTCC 5153

TCCTTCTTCA ATCCTTACAG TTTATGGCTC CACAGGAAGC TAATAAAGCG GGTAACAGGA 5213

CAACAAACCT TTATTACTGC CTTCAGAGAA TCTGTCCTCA CCTCCTGACC TCGACCCCTC 5273

ATCTAATCCA CAGATGTATG TCCACCATTG TTTTTTTTTC AGAGAACCAT TAAGTTCTGA 5333

CATAACCCAG TTTCTTTCAT TTACTATCTC AATGATCAAC GTTTAGCCGA TTCCAACAGT 5393

ATCTTTGGGT CTTTAACCCA TGTATTATTA CTATTATTGT TCATTGATCA AGACTGTTCT 5453

TGAGAAGTAT GGTGACCTAG AACACACACA TTAAAATGTG TCAACTATAA CCCATTCTTC 5513

TTTTTTCCCC CCCGAG GTC GAG ACC TAC CTG ACC GTC GCC AAG TGC AGG 5562 Val Glu Thr Tyr Leu Thr Val Ala Lys Cys Arg 170 175

AAG TCA CTG GAG GCC AAC TGC ACT CTG TAGACGTGGG CTGGAGAGGC 5609

Lys Ser Leu Glu Ala Asn Cys Thr Leu 180 185

TGCCAGCAAG AGCCTATCTC CAGGGTTCGG TTTCCCAGAT ACAGATTAGG CCTTGCCCTG 5669

CACTGAGGTG CATTTTCAAT TGAGATTCTC CATTAAACAT GCTTTTCAGT CTAGAGTAAT 5729

TTTATTTTGG ATCTGGTAGA GCCTGACTCC AGGGGTTTTC AGGCATTTGC ATTTTTTTCT 5789

CTGAAATCAA TAACAACACT TTCTATATTG ACTCTATCAC TCTGAGCTAC CATTGATTAG 5849

TACATTTATA TTAAAGGTTA TTAAATGTCT TATTTAGATA TATGGTTCAT GGCGGTGCTA 5909

CTTATGCATA CGTTAATATT TAGGGGTGAA ATGGGAACTT GTAGAGCTCC AAGCTTTTGG 5969

ATAATATATT TTAGAGTAAT TTCCTTTAAG TATTTTCATT CCTTAATCTT ATTGTTTGAA 6029

ACTAATAGTG ATTCATGTTT CAATAAAGAT GTTCTTCTCT GCAGCACATG ATCTCTTGGC 6089

TTCTATTTAA TATCTTTCAA ATCAACATTT TTTACAAGTT CCTAGCC 6136

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5474 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..5474

(D) OTHER INFORMATION: /standard_name= "Nucleotide

Secjuence of the Chinook Salmon GH Pseudogene"

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1349..1414

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1415..1810

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1811..1950

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1951..2086

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 2087..2203

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2204..2660

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 2661..2816

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 2817..4769

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 4770..4853

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 4854..5003

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 5004..5474

(i ) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 134..399

(D) OTHER INFORMATION: /note= "The 266 bp homologous region shared with GH-I in their 5' flanking regions. "

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 4836.-4838

(D) OTHER INFORMATION: /note= "The premature stop codon in exon 5."

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

AAGTCTAAAC AACAACTGAA CTTTTTGAGT GATTTCTGAC ATTTTGCTAC AGGCAATACA 60

TATCCCACAA TGGGGCCAGA TATGAGATGT TGGTTGATGG AATAAAATAT TTATTGATAC 120

ATGCTGTACA TGAAGTATTT TTGATTTGCT GGACTATCAA AACAAGTAAT CACTACATCC 180

AGTCAAATAG CCTGGCCAAA ACTACCCATT GTATAATGGT CTTTTGAATG CCAGCTATCA 240

CATATTCAAC AAGGTCATGT GGTCAGTAAA TGCACTCGCT GCCACCCTTC CAAAGTGACG 300

CAGGTTAACA GATCGATTTC TGTTTTCGCC AGGAAAGGAT TAGGACAGTG CTGGGCCTGA 360

CATTCAGCAC GTCTCCATGT CCTGAACGCT GAATATGGAC CTTGTTCACA ACTAGCTGTT 420

GAATATGTGC TCCCTGTATG CCACAACGAC AGACATCAAC CATGAACTCA TTACTCTCAA 480

TTGACCAGGT AACCTTTATA GTGGTCAAGA CACAGTATAC CTACACCTAC CATTGTAGCA 540

TAAACTCAAC ACAGTAAGGT TGTGCGGCAC AAACAGAGTG AAGGCTTTGG GCTAGTAGCG 600

CACATGCTGC TTGGCTGCAA GCATTCACGT GTTATCCACT GAGATGTGTA CTTTTTTTAA 660

AGGCATATAG ATTCTTACCC CAAAATCAAT CAATCAATCA ATGAAGTATG GCCTACACAC 720

TTGCTCCAAC AGATACCTTT ATTGGTCTTC GTCAGCTATA ACACAGGCTC TCACACAAAA 780

AGAAAATGTG GGTATTGATA TAGTGACTGT TTATGTTATT TAAACAAACT AAAAAACATA 840

CTTTGTAAGA TGTGTGTCTC CAGATAGTTC CTTCTCTGTG TGTCTACTTT GAGGTCTGTG 900

CTAACACCAT AGGACATTCA ATTGACATTA AACAATAAAA TATTGGTGCT AATAAAGAAG 960

CAATATAATA TATTTGTCAA ATAATGCCTG TTATTTACAG TACCGCGGAA CGGCAGAATA 1020

ACCGGTGTTG TTGTCAAGTT ACATGTCTGT CTGTGTGTGA GTGTAACTTT TGTTCATTCA 1080

TTATGTCCTA GACAACAGAG GTTTGTTTTG TATGTGTTTT GACCCTAATT TGTCAAGTAA 1140

GTTTTTTTGT AGGAGAGTCA CCAGTTCCTG AACTCATGGA AAAATTCATG ATTGATTTGA 1200

CGTATTATAC TGATTGTTCC ATAATGGCAT ACAAAAACAG GTTACATCAG CGACAGGTGG 1260

TAAATGGCGA AAATCTCATG TTTACTCATG TTGATACATT AAAACATATG TTCCCCATCT 1320

ATAAAAACAG TGGCCGCAAA CGAAGGGCTA ACCACATCAG CCACCACACT GTCAAGTCAT 1380

CATCCTTGGC AATTAAGAGA AGAAATCTGA CTTGGTAAAC CAGCTTTTAT TTTCCTTTTT 1440

TAAGTGAGAA GTCAGTGTAC CATTTAATAC CATTTAACTT TAACATTTAA ACATTTAACA 1500

TTAACATTAC TAGTTACAAC CTAACTATGT GATCTATTAG ATTTACATTT TAGTTATTTA 1560

GCAGACAGTC TTATCCAGAG TGACTTACAG GAGCAATTAG GGTTAAGTGC CTTGCTCAAG 1620

GGCACATCAA CAGATTTCTC ACCTAGTCAA CTCAGGGATT CAAACCAGTA ACCTTTCAGT 1680

TACTGGCCCA ACGCTCTTAA TCGCTAGGCT ATTGATGTAC CAAGGCTGAG AATATCTTAC 1740

TAACATGTCG CAACATAATT TGACTTACTT GTTTTTATAC ATTGCTTATT TTCTTTCATC 1800

TGACTTTTAC TGTTTCTGCT GATGCCAGTC TTACTGTTCA GTTGTTTTCT GGGTCAAGGG 1860

GAAGTGATGG AAAACCAACA GCTCTTCAAC ATTGCAGTCA ACAGGGTGCA ACAGTTCCAC 1920

CTATTGGCTC AGAAAATGTC CAACGACTTT GTAAGACATA TTTTGAATCT TCTTTTGACA 1980

TAGCAGATCA TGTTTCAGAG GTGATTCTTC TTCTTGTAAA CAGTGTCCTC TTCACACAAA 2040

CCTAGCGGCA AAAAATTCTC TCTCCCTTCT GTGTGATTTT GTGCAGGAAG GCACCCTGTT 2100

TTCTGATGAA CGCAGACAGC TGAACAAGAT ATCCCAGCTG GACATCTATA ACTCTGACTT 2160

CATCATGAGC CCAATCGACA AGCAGGAGAC TCAGAAGACT TCAGTAAGTT ACCTGGCGGA 2220

GCAAATCCGC ACGATGCACG ATTCCAAAAT TAATAATAGG GCATCTCAAT TTGAATAATC 2280

GATACAACTT AGTCATTAGT TATTGGGCAA GCAGATCCCC GATTGGTCTA AACTCCATGG 2340

GTAAATATAT ACTGTAGGAA AGCAGAACCA GCATCATGCA TGGTGGAAAT TAAATCTAGC 2400

CATGATAGGG AGTTTTAAAT TGTACACTTA AAATCAGCCA GTAAAATGTT GCTATACCTC 2460

AGTGCCTTCA ATTAAGGTAG TTAAAACAAC CACACACCAT AGTCCTTGTA AGTAAAACTC 2520

ATCACTCTCT AATCGGCGGT TTCTCTACAT CTACATTCTC CAGCCATGTA TCATGTAAAT 2580

GATATGTCAT CTCAAGCTGT ACAATACAAT TCAACTTCAT TTTCTAATAA TCTGTGGTTT 2640

CTCTAC TCT ACACACACAG GTCCTTAATC TTCTCCATAT CTCTTTCCAC CTGATTGAAT 2700

CCTGGCAGTA CTCTAGCCAG ACCATGACCA TCTCAAACAG CCTGATGGTC AGAAACTCCA 2760

ACCAGATCTC GAAGAAGCTC AGTGACCACA AAGTGGGCAT CAACCTGCTC ATCAAGGTAA 2820

AGAAAGGAGG GAGAACAATG ACCATTGTGG TGCCGACTTT GTGCATTTAT AACTCAAATA 2880

CTTCTAGTAA GTTGAACTCA GTCAAGTCAT TATTAAAATG TCTATGTGCT ACTGGCTCAC 2940

ATCTAAATGA GTCACATCAA TGCAAGTTTT TAAAGTTATA ACAAATTAAC TTTTTACCCA 3000

GCATGCTCTA CTACAGGTAG ATTGTTTTTG GAATTGTTCT TAATATCTGT GTTTTTACAT 3060

GTACAGTACA TTGCTGGCTG CCGCTCCAGC CCACGTTTAC ACTACATTGA GTGATTGATT 3120

AGATTATTAA TCTTATGCTA CGGAATCTAA AATAAAAATC CCGAAAATCA TATTGTATCA 3180

TTTTTAAGTC ATTCATTTGC AGTTCATTGC ATGACATAAG AATTTGATCA CCTACCAACC 3240

AGTAAGAATT ACGGCTCTTA CAGACCTATA TTTTTATATT GTCTTTGCAA TGGGATATTT 3300

CATAGGAACC AATTTTTTTC CCCTACCGCT TCCGCTGGAT GTTGCCAGTC TTTGGAATTT 3360

GGTTGAGGTT ATTTCTTTGT GCCATGAAGA AGTAGGACAA CTCGGAACTG GGGACACTTT 3420

TGTGAGTTGA GCAAGATGTG AAATCCAGTG CTGGTTTCTT TTCTTTCCTG TACAGATTGC 3480

CGTCTACAAT TTGATTGATT ATTAACATTT AAAAATACCC AACGTTGTAT TACAAAAGTA 3540

GTTTGAAATA TTTTGGCAAA TTTTATAAGA CAACTTTTGA AATATTTTGT AGCGATGTTG 3600

CGTTTTTGTA AGCTGTCTTT TTCTGGATCA AACGCTCTTT ATAAAGGGAC ATTTTCAATA 3660

TATATGGACG GAATTAATCA AACAAAAGGA CCAATTGTGA TGTTTATGGG ACATATTGGA 3720

GTGCCAACAA AAGAAGCTCG TCGAAGGTAA TGCATGTTTT TTATATTTTT ATTTCAGCGT 3780

TTTTGTGTAG CGCCTGCAGG GTTGTAATAT GCTAGCTCCT TTGTTTACTG CTGGTGCAGA 3840

TGGTACAGGC TATCAGATAA TAGCTTCTTA CTGTTTCGCC GAAAAGCAGT GTAAAAATCT 3900

GACATGTTAG CTGGATTCAC AACGAGTGTA TAGCTTTAAT TGAGTATCTT ACATGTGTGA 3960

TTTAATGAAA GTTTGAATCT TATAGCATTT ATTTGAATCT GGGTGCTCTG CATTTCCCCC 4020

CAGCAATTGG CCAGTTGAGA CGTTAGCATC TCGGCTATCT GTAAGAGTTA AGAATCCCTC 4080

CTGTTCTCCA CTCATACCTG TATTAACTGC ACCTGTTTGA ACTCATTACC TGTATACAAG 4140

ACACCTGTCC ACACACACAA TCAAACAGAC TCCAACCTCT CCACAATGGC CAAGACCAGA 4200

GGGCTGTGTA AGGGCATCAG GGTTACAAAT GTAGACCTGC ACAAGGCTGG GATGGGCTAC 4260

AGGAAAATAG GCAAGCAGCT TGGTGAGAAG GCAACAACTG TTGGCGCAAT TATTAGAAAA 4320

TGGAAGAAGT TCAAGATGAC GGTCAATCAC CCTTGGTCTG GGGCTCCATG CAAGATCTCA 4380

CCTCGTGGGG TATAAATGAT CATGAGGAAG GTGAGGGATC AGCCCAGTGT AGCTGCAGGA 4440

CCTGGTCAAT GACCCGAAGA GAGCTGGGAG CACAGTCTCA AAGAAAACCA TTAGTAACAC 4500

ACTACGCCGT CATGGATTAA AATCCTGCAG TGCATGCAAG GTCCCCCTGC TCAAGCCAGC 4560

GCATGTCCAG GCCCGTCTGA AGTTTTCCAA TTACCATCTG GATGATCCGG AGGGGAATGG 4620

GAGAAGGTCA TGTGGTCTGA TGAAACAAAA ATAGAGCTTT TTTGTCTAAA CTCCACTCGC 4680

TGTGTTTGGA GGAAGAAGAA GTATGAGTCG GGGATTTGTA TTGATATGCA AACATCCACC 4740

CCACCATGCA TCTCTCTCTG TCTCCACAGG GGAGCAAGGA TGGGATACTG ATCCTGGGTG 4800

ACAATGACTC TCAGCATCTG CCCCCCTGCG GGAACTAATA CCAGAACCTG GGGCGAACCT 4860

GGACGTATCT TTGGGTCTTT AATCCATATA TGATTTCTAT TATTGTTCAT TGATCAAGAC 4920

TGGTCTCGAG AAAGTCCTAG TGACATAGAA CATTCACATT AAAATGTGTC AAATATAACC 4980

TATTCTTCTT TTTTCCCCCC AAGGTTGAGA CCTACCTGAC CGTCGCTAAG TGCAGGCAGT 5040

CGCTGGAGGC CAACTGCACT CTGTAAACGT GGGCTGGAGC GGCAGCCAGC AAGAGCTGTC 5100

TCCAGGGTTC GGTTTCCCAG ATACAGATTA CACCTTGCTC TGCACTGAAG AGCATTTTCA 5160

ATTGAGATTC TCCATTATGC ATGCTTTTTA GTCTATAGTA GATTAATTAG TAGATTTAAT 5220

TTAGATCTGG TAGAGCCTGA CTCCAGGGGT TTTCAAGCAT TTGCATTTTC TTCTCTGAAA 5280

TCAACAACAG CACTTTCTAT AATATTCACT CTATTCCTCA GAGCTACCAT TGATCCACGG 5340

ACATTTTAGA TTAGTACATT TATAGAAAAG ATTATAAATA TGTCTTATTT AAATATATGA 5400

TTCGAGGTGG TGCTGCCATT TATGCATAAA TTAATATTTA GGGGTGAAAT GGGAACTTGT 5460

AGAGCTCCAA GCTT 5474

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 401 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..401

(D) OTHER INFORMATION: /standard_name= "Partial DNA Sequence of Chinook Salmon GH-II Gene" /note= "Sequence spans from the 3' end of exon 5, intron 5 and 5' end of exon 6."

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..124

(D) OTHER INFORMATION: /standard_name= "3' end of exon 5"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 125..339

(D) OTHER INFORMATION: /standard_name= "Intron 5"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 340..401

(D) OTHER INFORMATION: /standard_name= "5' end of exon 6"

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

CCTGGATGAC AATGACTCTC AGCATCTGCC CCCCTACGGG AACTACTACC AGAACCTGGG 60

GGGCGACGGC AACGTTAGGA GGAACTACGA ACTGTTGGCC TGCTTCAAGA AGGACATGCA 120

TAAGGTGGAA GATCACGTTG CCTTCAATTG CATGTGCCTT CCTATATTTT CTACGGTGCA 180

TTGTTTTTTT TGTAATCTCT ATTGTGAAGC CTTTGGGTCT TCAACCCATA TGTTATTACT 240

ATTATTGTTT ATTGATCAAG ACTGGTCTCG AGAAAGTCCT GGTGACTTAG AACATGCACA 300

TTAAAATGTG TCACTATAAC CTATTCTTCT TGTCCCAAGG TTGAGACCTA CCTGACCGTC 360

GCTAAGTGCA GGAAGTCACT GGAGGCCAAC TGCACTCTGT A 401

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCTGGATGAC AATGACTCTC A 21

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTACAGAGTG CAGTTGGCCT C 21

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GAAAATGTTC AATGACT 17

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Vi) ORIGINAL SOURCE:

(A) ORGANISM: Chinook Salmon

( i) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCTATCACT CTGAGCT 17