Abstract:
Killifish genes which are expressed specifically to females in accordance with the phenotype sex, characterized by having a base sequence selected from among the base sequences represented by SEQ ID NOS: 1 to 21 in Sequence Listing.

Description:
TECHNICAL FIELD  
         [0001]    The present invention relates to a method for rapidly assessing an endocrine-disrupting activity of a chemical substance as well as a series of techniques connected therewith in fields of medicine, pharmacology, environmental study and sitology.  
         BACKGROUND ART  
         [0002]    Endocrine disruptors (often called environmental hormones) collectively refer to chemical substances released into the environment for which hormone-like activities or anti-hormonal activities have been found. Altered reproductive potential (in particular, conversion of male into female), decreased reproductive potential, decreased hatchability, decreased survival rate of offspring, abnormal reproductive behavior and the like have been reported to be resulted from the influences of endocrine disruptors on the ecosystem of wild animals. Decreased number of sperms, endometriosis, infertility, ovarian cancer, uterine cancer, prostatic cancer and the like have been suspected to be resulted from the influences of endocrine disruptors on human health, although they have not been demonstrated.  
           [0003]    Substances (or groups of substances) that are considered to cause endocrine disruption are reported in the interim report (July 1997) by “Exogenous Endocrine Disrupting Chemical Task Force” of Environment Agency. However, it is considered that the types of such substances would be further increased in the course of research and study in the future.  
           [0004]    Known methods for determining endocrine disrupting activities are classified into two groups, i.e., in vitro methods and in vivo methods. Examples of the methods in the former group include a method in which an activity of binding to estrogen receptor, androgen receptor or thyroid hormone receptor is measured, and a method in which an activity of inhibiting a hormone synthesis enzyme system is measured. Examples of the methods in the latter group include a method in which production of various hormones and abnormal tissue formation in individuals at different postnatal days are determined, a method in which abnormal metamorphosis in a frog is determined, and a method in which abnormal maturation in a fish is determined (Analytical Chemistry, 70(15):528A-532A (1998)).  
           [0005]    The in vitro method is advantageous because it is sensitive, and it can be used to assay a number of test samples in a short time. However, it cannot be determined whether or not endocrine is actually disrupted using the in vitro method because it only determines an activity of binding to a receptor or the like. On the other hand, the actual influence on endocrine is directly examined using the in vivo method in which animals such as rats, frogs, fishes or the like are used. Thus, such a method is necessary in order to determine the influence on a living body or the environment. However, the in vivo method has drawbacks because, for example, its sensitivity is low, it requires complicated operation and, if a number of samples are to be examined, it requires a long time.  
           [0006]    For example, a system in which conversion of male into female is monitored to assess an endocrine-disrupting activity has been constructed. The monitoring is carried out by determining the expression of a fish female-specific yolk precursor protein vitellogenin in males using an anti-vitellogenin antibody. However, it is difficult to deal with a number of test samples at a time using the system. The sensitivity of the system in which adult fishes are used for the assessment is supposed to be lower than that of an assessment system in which fries are used because fries are relatively subject to disrupting activities. The system has further problems because it requires a wide space for breeding and a long breeding period. In addition, anti-vitellogenin antibodies specific for the respective fishes to be assessed are required for the system.  
           [0007]    An assessment method in which the hatchability of eggs or the number of eggs spawned from an adult fish are used as indexes, and an assessment method in which courtship behavior or the like is monitored have been proposed (Lisa, D. et al., Environmental Toxicology and Chemistry, 17:49-57, 1998). However, it cannot be determined whether or not sexual differentiation is actually disrupted using such methods.  
           [0008]    Disruption of sexual differentiation can be examined by determining both the genotypic sex and the phenotypic sex of an individual and comparing them each other. For example, a system for assaying a disrupting activity in which sex reversal is used as an index has been proposed. In the system, a fry or an egg is bred while administering an environmental hormone thereto, and the phenotypic sex associated with secondary sex characteristics is then determined. For example, in case of medaka, the genotypic sex can be determined by using a PCR (Shinoyama, A. et al., The Fish Biology Journal MEDAKA, 10:31-31, 1999), or a medaka strain of which the genotypic sex is linked to pigment expression, d-rR (Yamamoto, T., J. Exp. Zool., 123:571-594, 1958) or Qurt (Wada, H. et al., Zoological Science, 15:123-126, 1998). It is required to prepare a tissue section to microscopically examine a gonad in order to determine the phenotypic sex of a fry. Thus, it is difficult to deal with a number of test samples. Breeding for at least one month is required in order to determine the phenotypic sex based on the shape of a fin associated with secondary sex characteristics. Furthermore, skill is required for the determination. It is also difficult to deal with a number of test samples using this method. As described above, in fact, the in vivo assay requires a long time from the start of assessment, and it is difficult to assay a number of test samples at a time. However, such an in vivo assay is necessary for assessing chemical substances or water environment, or monitoring water pollution. In addition, construction of a rapid and accurate assay system has been desired.  
           [0009]    Examination of an endocrine-disrupting activity may provide an index for assessing the influence of a chemical substance on humans, for determining the influence on living bodies upon its release into the environment or on the ecosystem, or for monitoring water pollution. However, the prior art has drawbacks as described above. Thus, a sensitive and rapid method for determining an endocrine-disrupting activity has been desired.  
         SUMMARY OF THE INVENTION  
         [0010]    As a result of intensive studies, the present inventors have successfully isolated and identified genes expressed specifically in phenotypic females of medaka during early development for the first time. The present inventors have found that disruption of sexual differentiation can be examined by rapidly determining the phenotypic sex for a fry of medaka using the gene, and comparing the phenotypic sex with the genotypic sex. The present inventors have also found that an endocrine-disrupting activity of a sample can be rapidly determined using the method. The present inventors have successfully constructed a method for rapidly assessing endocrine-disrupting activities of chemical substances, samples of water environment (lakes, marshes, rivers, seas, etc.) or the like in vivo for a number of samples at a time. Thus, the present invention has been completed.  
           [0011]    The present invention is outlined as follows. The first aspect of the present invention relates to a gene expressed in a female-specific manner depending on its phenotypic sex, which has a nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOS: 1 to 21. It also relates to a gene which hybridizes with said sequence under stringent conditions as well as a gene which has said sequence as well as an intron or introns being inserted.  
           [0012]    The second aspect of the present invention relates to a method for assessing a sexual differentiation-disrupting activity of a sample, the method comprising:  
           [0013]    (1) administering a sample to be assessed for its sexual differentiation-disrupting activity to a medaka;  
           [0014]    (2) determining the genotypic sex of the medaka;  
           [0015]    (3) determining the phenotypic sex of the medaka based on the expression of a female-specific gene; and  
           [0016]    (4) determining if the sexual differentiation is disrupted based on the results of steps (2) and (3).  
           [0017]    The third aspect of the present invention relates to a method for detecting an endocrine disrupter, comprising assessing a sexual differentiation-disrupting activity by the method of the second aspect.  
           [0018]    The fourth aspect of the present invention relates to an oligonucleotide for detecting the gene of the first aspect.  
           [0019]    The fifth aspect of the present invention relates to a kit for assessing a sexual differentiation-disrupting activity by the method of the second aspect.  
           [0020]    The sixth aspect of the present invention relates to a kit for detecting an endocrine disrupter by the method of the third aspect.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0021]    The present invention is described in detail below.  
           [0022]    The present invention may be applied to any samples without limitation, including naturally occurring or artificially synthesized chemical compounds. Such a substance may be subjected to the method of the present invention in an isolated form or in a mixture. The method of the present invention is applicable to a sample from the environment such as river water, soil or the like.  
           [0023]    A medaka ( Oryzias latipes ) is used as an organism according to the method of the present invention. It is widely used as a material for studying genes because it is small and easy to handle, it releases a lot of eggs upon spawning, its generation time is short (about three months), and a genetically homogeneous strain can be established by inbreeding.  
           [0024]    A medaka can be bred in distilled water in a 96-well microplate for about one week after hatching. Since feeding is unnecessary, secondary factors such as a disrupting activity due to a bait can be excluded upon assessment.  
           [0025]    One can breed a medaka in water containing salt at various concentrations, including fresh water and seawater, at a wide range of temperatures from about 0 to about 30° C. Thus, it can be used for assessment assuming various environments.  
           [0026]    There is no specific limitation concerning the medaka to be used according to the present invention. A wild type medaka or a medaka strain of which the genotypic sex is linked to pigment expression such as d-rR or Qurt may be used.  
           [0027]    Qurt is a medaka strain heterozygous for the leucophore free (if) locus, which is closely linked to sex. A female (X lf /X lf ) individual of Qurt is colorless, whereas a male (X lf /Y + ) individual is yellow as a result of pigment expression. The yellow color can be observed for an egg. Therefore, Qurt can be preferably used according to the present invention because its genotypic sex can be readily determined by microscopically examining the egg without extracting the DNA (Zoological Science, 15:123-126, 1998).  
           [0028]    There is no specific limitation concerning the method for determining the genotypic sex according to the present invention. For example, the genotypic sex can be determined by genetic analysis of sex chromosomes.  
           [0029]    The genotypic sex of medaka is fixed upon fertilization depending on the combination of sex chromosomes as follows: female in case of X/X; and male in case of X/Y. Thus, the genotypic sex can be determined by genetic analysis of sex chromosomes. There is no specific limitation concerning the method for the genetic analysis. For example, the analysis can be carried out by hybridization using a probe that hybridizes with a gene on the sex chromosomes or a PCR using primers that can be used to amplify a gene on the sex chromosomes. If such a method is used to determine the genotypic sex, it is also necessary to prepare both DNA and RNA from an individual. This is because it is necessary to analyze the expression of a female-specific gene using RNA for determining the phenotypic sex as described below. In this case, the head portion of a fry is cut off. The remaining body portion which contains a gonad and the like is used to determine the expression of a gene that is specifically expressed depending on the phenotypic sex as described below. DNA extracted from the head portion is used to determine the genotypic sex. Alternatively, DNA and RNA may be prepared simultaneously using QIAGEN RNA/DNA System (QIAGEN). Furthermore, DNAs can be prepared simultaneously from a number of test samples in a 96-well microplate using DNeasy 96 Tissue Kit (QIAGEN).  
           [0030]    If the medaka strain Qurt is used, the difference in pigment expression specific for the genotypic sex can be recognized on the second day after fertilization. Thus, the genotypic sex can be determined for an egg by detecting the pigment without preparing DNA.  
           [0031]    A gene that is expressed in a medaka in a phenotypic sex-specific manner is used for determining the phenotypic sex according to the method of the present invention. For example, a medaka gene expressed in a phenotypic female-specific manner is used. A gene that is specifically expressed within five days after hatching is preferable for assessment at an early stage.  
           [0032]    Examples of such genes include the following:  
           [0033]    (1) FIGα, a transcription factor containing a basic helix-loop-helix motif;  
           [0034]    (2) eIF-4, a cap-binding subunit of an elongation initiation factor;  
           [0035]    (3) genes encoding ZP domain-containing proteins;  
           [0036]    (4) 42Sp50 and 42Sp43, genes encoding oocyte-specific RNA storage proteins;  
           [0037]    (5) quinone reductase gene; and  
           [0038]    (6) unknown genes encoding secretory proteins.  
           [0039]    Such genes are exemplified by ones having the sequences of SEQ ID NOS: l to 21 or sequences that hybridize with said sequences under stringent conditions. Stringent hybridization conditions include, for example, those as described in T. Maniatis et al. (eds.), Molecular Cloning: A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory, 1989. Incubation with a probe at 65° C. overnight in a solution containing 6×SSC (1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5× Denhardt&#39;s and 100 mg/ml herring sperm DNA exemplifies the conditions.  
           [0040]    The gene may exist on a chromosome with an intron or introns being inserted. The present invention also encompasses such a gene having an intron or introns being inserted. Examples of such genes include a gene having the nucleotide sequence of SEQ ID NO: 22 (the sequence of SEQ ID NO: 1 with introns being inserted) or a gene having the nucleotide sequence of SEQ ID NO: 23 (the sequence of SEQ ID NO: 8 with introns being inserted).  
           [0041]    For example, the above-mentioned genes can be isolated as follows.  
           [0042]    The sexual differentiation of a medaka is first manifested as a phenomenon that the number of germ cells in a female is about twice as many as that in a male upon hatching, i.e., on about tenth day after fertilization (Satoh, N., Egami, N., J. Embryol. Exp. Morph., 28:385-395, 1972). This is because mitosis is initiated immediately after hatching in a portion of germ cells in a female whereas germ cells in a male do not divide until two months after hatching. It is possible to identify genes expressed in a female-specific manner at an early development stage using the difference in sexual differentiation in germ cell line as an index. Difference in gene expression between a male and a female of medaka can be examined using subtractive hybridization. Genotypic male and female are separated each other before hatching. RNAs are extracted from the both after hatching. Then, a gene expressed in a female-specific manner can be isolated using subtractive hybridization. If the medaka strain Qurt is used, genotypic male and female can be readily distinguished before hatching on the second day after fertilization using the expression of the pigment gene as an index.  
           [0043]    A genomic gene corresponding to each gene can be isolated by screening a genomic library according to a known method using the thus obtained gene as a probe.  
           [0044]    The gene can be detected using an oligonucleotide designed based on the nucleotide sequence of the gene. The oligonucleotides for detecting the gene according to the present invention include, but are not limited to, primers that can be used to amplify the gene or a portion thereof according a gene amplification method, and a probe that is hybridizable with the gene under stringent conditions.  
           [0045]    Examples of gene amplification methods that can be used include, but are not limited to, PCR, SDA, NASBA and ICAN (WO 00/56877).  
           [0046]    A primer or a probe can be designed at will based on the nucleotide sequence of the gene. Of course, a sequence is selected upon designing such that the primer or the probe does not form a secondary structure within the molecule, and attention is paid such that the melting temperature (Tm value) for the primer or the probe and the corresponding template is set at an appropriate temperature.  
           [0047]    The Tm value of a primer or a probe can be determined, for example, according to the following equation:  
           Tm=81.5−16.6(log 10 [Na + ])+0.41(% G +C)−(600/N)  
           [0048]    wherein N is the chain length of the primer or the probe; % G+C is the content of guanine and cytosine residues in the primer or the probe.  
           [0049]    If the chain length of the primer or the probe is shorter than 18 bases, the Tm value can be estimated, for example, as the sum of the product of the number of adenine and thymine (A+T) residues multiplied by 2(° C.) and the product of the number of G+C residues multiplied by 4(° C.), i.e., [(A+T)×2+(G+C)×4].  
           [0050]    Although is not intended to limit the present invention, the chain length of the probe is preferably 15 bases or more, more preferably 18 bases or more in order to avoid nonspecific hybridization.  
           [0051]    Although it is not intended to limit the present invention, for example, a primer of 15 to 40 bases in length can be used. In particular, a primer of 17 to 30 bases in length can be preferably used.  
           [0052]    Furthermore, it is desirable to design a primer such that the ratio of cytosine (C) and guanine (G) around the 3′-terminus becomes high. A commercially available software for primer designing such as OLIGO™ Primer Analysis software (Takara Shuzo) may be used for designing a primer.  
           [0053]    The primer or the probe may have a mutation such as deletion, substitution, insertion or addition of a nucleotide (or nucleotides) in a portion of the sequence as long as it can be used to detect the gene. In case of a primer, it is preferable not to include a mutation or, if any, to minimize mutations around the 3′-terminus of the primer because such a mutation greatly influences the efficiency of primer extension reaction. An appropriate sequence unrelated to the nucleotide sequence of the gene (e.g., a promoter sequence recognized by an RNA polymerase) may be added on the 5′ side of the primer. optionally, the primer or the probe may be appropriately modified. Addition of a ligand such as biotin or digoxigenin, or a fluorescent substance to a primer or a probe facilitates the detection of the amplification reaction product.  
           [0054]    A product of a gene amplification reaction using the primers can be detected by subjecting a portion of the reaction mixture after the amplification reaction to electrophoresis, and then staining the DNAs with ethidium bromide. An amplification product can be detected without electrophoresis by utilizing hybridization. If a modified primer is used, a detection method suitable for the modification can be used.  
           [0055]    A kit containing the primer or the probe can be constructed and used for detecting the gene according to the present invention. Such a kit may contain a buffer or an enzyme to be used for an amplification reaction or hybridization. It may further contain a reagent to be used for preparation of a nucleic acid sample from cells or detection of an amplification product in order to make the detection more convenient.  
           [0056]    There is no specific limitation concerning the method for detecting the expression of the gene. For example, the expression can be detected by detecting the mRNA transcribed from the gene in RNA prepared from a medaka on the 1st to 5th day after hatching by Northern hybridization, RT-PCR or the like.  
           [0057]    RNA can be prepared from a medaka, for example, by directly treating the medaka individual using TRIzol reagent (Gibco-BRL) or the like. Alternatively, RNA and DNA can be simultaneously prepared using QIAGEN RNA/DNA System (QIAGEN). In this case, the DNA can be used for the determination of the genotypic sex. Furthermore, DNAs can be prepared simultaneously from a large number of test samples in a 96-well microplate using RNeasy 96 Kit (QIAGEN).  
           [0058]    In order to exclude false positive results due to products amplified from a genomic DNA, it is desirable to use a pair of primers for RT-PCR that can be used to amplify a region of mRNA transcribed from the gene of interest and that is designed such that the corresponding region in the gene contains an intron or introns being inserted. For example, a combination of a primer F1 (SEQ ID NO: 30) and a primer R1 (SEQ ID NO: 31), a combination of a primer F2 (SEQ ID NO: 32) and a primer R2 (SEQ ID NO: 33) or the like can be used to detect Gene 5. The primers F1, R1, F2 and R2 have sequences in the exons 2, 8, 2 and 7, respectively.  
           [0059]    If the primers F1 and R1 are used, the size of the product amplified from the mRNA is about 730 bp, whereas the size of the product amplified from the genomic DNA is about 1.1 kbp. If the primers F2 and R2 are used, the size of the product amplified from the mRNA is about 590 bp, whereas the size of the product amplified from the genomic DNA is about 0.9 kbp. Thus, the product amplified from the mRNA can be clearly distinguished from the product amplified from the genomic DNA as a background.  
           [0060]    More sensitive detection can be accomplished using these primer pairs by carrying out a 1st PCR using the pair of primers F1 and R1 followed by a nested PCR using the pair of primers F2 and R2.  
           [0061]    A combination of a primer 863.3 (SEQ ID NO: 24) and a primer 863.1 (SEQ ID NO: 25), a combination of a primer 863.3 (SEQ ID NO: 24) and a primer {fraction (1/15)} (SEQ ID NO: 26) or the like can be used to detect Gene 1. The primers 863.3, 863.1 and {fraction (1/15)} have sequences in the exons 1, 3 and 4, respectively. If the primers 863.1 and 863.3 are used, the size of the product amplified from the mRNA is about 300 bp, whereas the size of the product amplified from the genomic DNA is about 1 kbp. If the primers 863.3 and {fraction (1/15)} are used, the size of the product amplified from the mRNA is about 400 bp, whereas the size of the product amplified from the genomic DNA is about 4 kbp. Thus, the product amplified from the mRNA can be clearly distinguished from the product amplified from the genomic DNA as a background.  
           [0062]    A combination of a primer 6a (SEQ ID NO: 27) and a primer 6b (SEQ ID NO: 28), a combination of a primer 6a (SEQ ID NO: 27) and a primer 8.3 (SEQ ID NO: 29) or the like can be used to detect Gene 8. The primers 6a, 6b and 8.3 have sequences in the exons 3, 7 and 8, respectively. If the primers 6a and 6b are used, the size of the product amplified from the mRNA is about 530 bp, whereas the size of the product amplified from the genomic DNA is about 1.3 kbp. If the primers 6a and 8.3 are used, the size of the product amplified from the mRNA is about 880 bp, whereas the size of the product amplified from the genomic DNA is about 2.2 kbp. Thus, the product amplified from the mRNA can be clearly distinguished from the product amplified from the genomic DNA as a background.  
           [0063]    In order to exclude false positive results due to a genomic DNA, it is desirable to use a probe for Northern hybridization that hybridizes with a region of mRNA transcribed from the gene of interest and that is designed such that the corresponding region in the gene contains an intron or introns being inserted.  
           [0064]    The expression of the gene can be detected using a DNA microarray. A DNA microarray is a material having nucleic acids being immobilized in which a number of different genes or DNA fragments are arrayed and immobilized on a solid phase substrate such as a slide glass. The DNA microarray is used to examine the existence of a nucleic acid in a nucleic acid sample that has a sequence complementary to the DNA immobilized on the microarray by contacting it with a nucleic acid sample (preferably a labeled nucleic acid sample) prepared from a sample for hybridization. Expression of plural genes specifically expressed depending on the phenotypic sex can be monitored at the same time using the microarray.  
           [0065]    Also, the expression of the gene can be detected using an antibody to a protein translated from the gene. There is no specific limitation concerning the antibody used as long as it can recognize the protein expressed from the gene. A polyclonal antibody, a monoclonal antibody or the like prepared according to a known method may be used.  
           [0066]    A sexual differentiation-disrupting activity of a sample can be assessed by comparing the phenotypic sex with the genotypic sex both determined as described above. An endocrine-disrupting activity of a sample can be assessed according to this method. Although it is not intended to limit the present invention, disruption of sexual differentiation may be represented by expression of a phenotypic female-specific gene in a genotypic male individual, or loss of expression of a phenotypic female-specific gene in a genotypic female individual.  
           [0067]    For example, a sexual differentiation-disrupting activity of a sample can be assessed as follows. A medaka egg immediately after fertilization is bred in a test water sample. The genotypic sex and the phenotypic sex are determined for each individual as described above. An individual is determined to be influenced by an sexual differentiation-disrupting activity if the genotypic sex of the individual is different from the phenotypic sex of the same individual. The sexual differentiation-disrupting activity of the sample can be assessed by counting the number of such individuals. If a medaka strain Qurt of which the genotypic sex is linked to pigment expression is used, the relationship between the genotypic sex and the pigment expression may be reversed due to translocation of chromosome with the probability usually at several percentage or less. This problems can be solved by increasing the number of test samples. Translocation of chromosome is rarely observed for a medaka strain d-rR of which the genotypic sex is also linked to pigment expression. Thus, almost no reversion is observed for the relationship between the genotypic sex and the pigment expression if this medaka strain is used.  
           [0068]    A kit for assessing a sexual differentiation-disrupting activity according to the present invention is one for assessing the activity using the method of the present invention. Although it is not intended to limit the present invention, a kit containing an oligonucleotide that can be used to detect the gene of the present invention as described above is exemplified. Such a kit may contain a buffer or an enzyme to be used for an amplification reaction. It may further contain a reagent to be used for preparation of a nucleic acid sample from cells or detection of an amplification product in order to make the detection convenient.  
           [0069]    A kit for detecting an endocrine disruptor according to the present invention is one for detecting an endocrine disrupter using the method of the present invention. Although it is not intended to limit the present invention, a kit containing an oligonucleotide that can be used to detect the gene of the present invention as described above is exemplified. Such a kit may contain a buffer or an enzyme to be used for an amplification reaction. It may further contain a reagent to be used for preparation of a nucleic acid sample from cells or detection of an amplification product in order to make the detection convenient.  
           [0070]    The phenotypic sex can be determined at an early stage by the fifth day after hatching according to the method of the present invention. Conventionally, the phenotypic sex could be determined only based on the shape change of a fin associated with secondary sex characteristics one month or more after hatching or the like. Furthermore, no special skill is required for the present invention. Thus, it is also possible to deal with a number of test samples according to the method of the present invention. The method is effective in rapidly and conveniently assessing endocrine-disrupting activities of naturally occurring or artificially synthesized chemical substances as well as samples from the environment such as river water or soil.  
       
    
    
     EXAMPLES  
       [0071]    The following examples further illustrate the present invention in detail but are not to be construed to limit the scope thereof.  
       Example 1  
       [0072]    Identification of Female-Specific Genes by Subtractive Hybridization  
         [0073]    A pair of mature Qurt medakas consisting of one male and one female (Zoological Science, 15:123-126, 1998) was bred in 3 L of green water (purified water containing chlorella from green algae) at 25° C. under conditions of a light period for 14 hours and a dark period for 10 hours. They were fed with TetraMin three to five times a day setting the amount of TetraMin such that it was consumed within three to five minutes, and then spawned. Embryos were genotypically classified into males and females by examining the expression of the yellow pigment gene as autofluorescence under a fluorescence microscope on the fourth day after spawning. mRNAs were prepared from samples at four stages (stage 37/39 (2-3 days before hatching), or 1, 5 or 30 day(s) after hatching) according the classification table of Iwamatsu (Iwamatsu, T., Zool. Sci., 11:825-839, 1994). cDNAs were prepared using an oligo(dT) primer and Copy Kit (Invitrogen). The cDNAs were cleaved with a restriction enzyme AluI, ligated to linkers (Wang, Z. and Brown, D., Proc. Natl. Acad. Sci. USA, 88:11505-11509, 1991), and amplified using PCRs. Subtractive hybridization was carried out using cDNAs from a male and a female at each stage. The remaining cDNAs were amplified using PCRS. This process of subtraction/PCR amplification was repeated three times. The amplified cDNAs for the males and the females at the respective stages were cloned into a plasmid to obtain eight cDNA libraries. Fragments inserted in clones selected at random from the respective libraries were isolated and subjected to Southern hybridization to screen for genes expressed in a sex-specific manner at each stage. The starting cDNAs and the cDNAs obtained after three rounds of subtraction/PCR amplification were used as probes for the Southern hybridization. No male-specific positive reaction was observed. Thus, no gene expressed in a male-specific manner could be isolated. Three clones and forty-seven clones were obtained from the 5th and 30th day female libraries, respectively, as clones that exhibit positive reactions in a female-specific manner. The fragments inserted in these clones were used as probes for hybridization with the cDNAs from the 1st, -5th or 30th day male or female (both the starting cDNAs and the cDNAs obtained after three rounds of subtraction/PCR amplification). Based on the results of hybridization, the genes were classified into three groups, i.e., groups of genes expressed in females on the 1st, 5th or 30th day. Nucleotide sequences of two genes classified as those expressed in females on the 1st day (Genes 1 and 2) and nineteen genes classified as those expressed in females on the 5th day (Genes 3-21) were determined. The determined nucleotide sequences of Genes 1-21 are shown as SEQ ID NOS: 1-21.  
         [0074]    Homology searches of database for the determined gene sequences revealed that the Genes shared homologies with known genes as follows: Gene 1 (SEQ ID NO: 1)—gene for FIGα, mouse transcription factor having a basic helix-loop-helix motif; Gene 2 (SEQ ID NO: 2)—gene for eIF-4, human cap-binding subunit of elongation initiation factor; Gene 3 (SEQ ID NO: 3)—gene encoding rabbit ZPA domain; Gene 4 (SEQ ID NO: 4)—gene encoding goldfish ZPB domain; Gene 5 (SEQ ID NO: 5)—gene encoding carp ZPC domain; Gene 6 (SEQ ID NO: 6)—gene encoding zebra fish ZPC domain; Gene 7 (SEQ ID NO: 7)—gene encoding carp ZPC domain; Gene 8 (SEQ ID NO: 8)—gene encoding zebra fish ZPC domain; Gene 9 (SEQ ID NO: 9)—gene encoding zebra fish ZPC domain; Gene 10 (SEQ ID NO: 10)—Xenopus 42Sp42 gene which encodes oocyte-specific RNA storage proteins; Gene 11 (SEQ ID NO: 11)—Xenopus 42Sp50 gene; and Gene 12 (SEQ ID NO: 12)—rat quinone reductase gene. Genes 13-21 (SEQ ID NOS: 13-21) did not share homology with a known gene. It is supposed that they unknown genes that encode secretory proteins based on the sequence characteristics.  
         [0075]    Next, a genomic library was constructed using chromosomal DNA prepared from a medaka according to a known method. Screening was carried out using Gene 1 or 8 as a probe. Corresponding genomic genes were isolated and the nucleotide sequences were determined. Nucleotide sequences of Genomic Gene 22 (corresponding to Gene 1) and Genomic Gene 23 (corresponding to Gene 8) are shown as SEQ ID NOS: 22 and 23.  
       Example 2  
       [0076]    Detection of Sexual Differentiation-Disrupting Activity of 17 β-estradiol  
         [0077]    Pairs of mature Qurt medakas each consisting of one male and one female were bred in 3 L of green water (purified water containing chlorella from green algae) at 25° C. under conditions of a light period for 14 hours and a dark period for 10 hours. They were fed with TetraMin three to five times a day setting the amount of TetraMin such that it was consumed within three to five minutes,;and then spawned. Eggs resulted from five pairs were placed in a Petri dish immediately after spawning, separated each other using tweezers in tap water from which chlorine had been removed and washed. The water was replaced by a 1 -ppb 17 β-estradiol (E2) aqueous solution containing 0.1% dimethyl sulfoxide. The mixture was dispensed into wells of 96-well round bottom microplate (#3797, Corning) which had been extensively washed with ultrapure water such that each well contained one egg. After covering with a lid, the microplate was incubated in an incubator at 25° C. On the third day from the start of incubation, males and females were distinguished by examining the yellow pigment which is expressed in a male-specific manner as autofluorescence using a fluorescence microscope (Nikon) The incubation was further continued, and fries then hatched on the 7th to 9th day after spawning. Fries were collected on the 12th day after spawning (the 3rd to 5th day after hatching), then soaked in RNAlater (#7021, Ambion) and stored at −80° C. RNAs were extracted from the fries using StrataPrep Total RNA Miniprep Kit (#400711, Stratagene). The test samples were soaked in a lysis solution attached to the kit, and homogenized in 1.5 -mL tubes using a pellet mixer (Urin Seisakusho). Then, extraction was completed according to the manual attached to the kit. The extracted RNAs were subjected to TaKaRa One Step RNA PCR Kit (AMV) (Takara Shuzo). 1st PCRs were carried out using a pair of primers F1 (SEQ ID NO: 30) and R1 (SEQ ID NO: 31) which is used to amplify a region of 729 bp in Gene 5 (SEQ ID NO: 5), a gene expressed in a female-specific manner. Next, nested PCRs were carried out using a pair of primers F2 (SEQ ID NO: 32) and R2 (SEQ ID NO: 33) which is used to amplify a region of 593 bp within the 729 -bp region using 0.5 μL each of the products of the 1st PCRs as templates. The resulting amplification products were subjected to electrophoresis on 2% agarose gel. The results are shown in Table 1. In the table, the genotypic sex represents the result of microscopic examination, and the phenotypic sex is represented as the result of distinction between a female and a male using the RT-PCR.  
                                                                                                             TABLE 1                           Control group       (treatment with solvent (water containing 0.1% DMSO))            Sample No.   1   2   3   4   5   6   7   8           Genotypic sex   ♂   ♂   ♂   ♂   ♀   ♀   ♀   ♀       Expression of Gene 5   −   −   −   −   +   +   +   +            Treatment with 1 ppb 17 β-estradiol            Sample No.   1   2   3   4   5   6   7   8   9   10       Genotypic sex   ♂   ♂   ♂   ♂   ♂   ♀   ♀   ♀   ♀   ♀       Expression of Gene 5   −   −   −   +   +   −   −   +   +   +                  
 
         [0078]    For the fries hatched from eggs treated with a solvent (water containing 0.1% DMSO) in the control group, the expression of the female-specific gene, Gene 5 was observed only for the genotypic females, and not for the genotypic males. Thus, the genotypic sex was consistent with the phenotypic sex. On the other hand, for the fries hatched from eggs treated with 1 ppb 17 β-estradiol, the expression of Gene 5 was observed for two out of the five genotypic males. Furthermore, the expression of Gene 6 was not observed for two out of the five genotypic females. These results show that the sexual differentiation was disrupted.  
       INDUSTRIAL APPLICABILITY  
       [0079]    The present invention provides medaka genes expressed in a female-specific manner depending on the phenotypic sex for the first time. The present invention provides a rapid and convenient method for determining the phenotypic sex using the expression of the gene as an index. The phenotypic sex can be determined within five days after hatching according to the method of the present invention. Conventionally, the phenotypic sex could not be determined until one month after hatching or the like. In addition, determination can be carried out for a number of test samples in a short time according to the method of the preset invention.  
         [0080]    A sexual differentiation-disrupting activity of a sample can be determined rapidly and conveniently by administering a sample suspected to have a sexual differentiation-disrupting activity to a medaka, determining the phenotypic sex of the medaka according to the method of the present invention and comparing the result of the determination with the genotypic sex.  
         [0081]    Sequence Listing Free Text  
         [0082]    SEQ ID NO: 1: cDNA for gene 1  
         [0083]    SEQ ID NO: 2: cDNA for gene 2  
         [0084]    SEQ ID NO: 3: cDNA for gene 3  
         [0085]    SEQ ID NO: 4: cDNA for gene 4  
         [0086]    SEQ ID NO: 5: cDNA for gene 5  
         [0087]    SEQ ID NO: 6: cDNA for gene 6  
         [0088]    SEQ ID NO: 7: cDNA for gene 7  
         [0089]    SEQ ID NO: 8: cDNA for gene 8  
         [0090]    SEQ ID NO: 9: cDNA for gene 9  
         [0091]    SEQ ID NO: 10: cDNA for gene 10  
         [0092]    SEQ ID NO: 11: cDNA for gene 11  
         [0093]    SEQ ID NO: 12: cDNA for gene 12  
         [0094]    SEQ ID NO: 13: cDNA for gene 13  
         [0095]    SEQ ID NO: 14: cDNA for gene 14  
         [0096]    SEQ ID NO: 15: cDNA for gene 15  
         [0097]    SEQ ID NO: 16: cDNA for gene 16  
         [0098]    SEQ ID NO: 17: cDNA for gene 17  
         [0099]    SEQ ID NO: 18: cDNA for gene 18  
         [0100]    SEQ ID NO: 19: cDNA for gene 19  
         [0101]    SEQ ID NO: 20: cDNA for gene 20  
         [0102]    SEQ ID NO: 21: cDNA for gene 21  
         [0103]    SEQ ID NO: 22: Genomic DNA for gene 1  
         [0104]    SEQ ID NO: 23: Genomic DNA for gene 8  
         [0105]    SEQ ID NO: 24: PCR primer 863.3  
         [0106]    SEQ ID NO: 25: PCR primer 863.1  
         [0107]    SEQ ID NO: 26: PCR primer {fraction (1/15)} 
         [0108]    SEQ ID NO: 27: PCR primer 6a  
         [0109]    SEQ ID NO: 28: PCR primer 6b  
         [0110]    SEQ ID NO: 29: PCR primer 8.3  
         [0111]    SEQ ID NO: 30: PCR primer F1  
         [0112]    SEQ ID NO: 31: PCR primer R1  
         [0113]    SEQ ID NO: 32: PCR primer F2  
         [0114]    SEQ ID NO: 33: PCR primer R2  
         [0115]    [0115] 
     
       
       
         1 
         
           
             33  
           
           
             1  
             1099  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 1  
             
           
            1 

gtaaacggac agctagagtt cagctgaaaa gaagtgtaat tctattcgga cgtagtcttt     60 

aaaaaaaaaa tgaaggtgcc agaggcggaa ttaatgagcg acattctgaa gaggctgacg    120 

ggagagtctg ctctgccgct gtactgctgc atcgagaagt acaagcgcga gaggaacggc    180 

ctctactttg tcgccgagga tttcactgaa accgtcaaaa aaagagaaat ggtcaacgcc    240 

aaggaaagac tgagaataag gaacttgaac acaatgttct cccgactgaa gcgcatgctg    300 

cctctaatgc aaccagacaa aaagccgagt aaagttgata cactcaaagc agccactgaa    360 

tacattcgac ttcttcttgc tgttttgcgg gacactgaaa ataacaacac tgggacggat    420 

tttctaaaga atgcaatcac ttatggtcag caggatggct tcgccaatga cctctggaga    480 

atggacgatt tcttgaacct gtcagatgat cacatggagg atgggttcac catgccagca    540 

gaacctgcag cagaggatgg agacatgact agactggtgt tgcagcattg tgtgatgcct    600 

gcataccagt tcatcatcca agtagcgccc gatcaagctt cgagggatta attagccacc    660 

gcctcccgac tgcacatccc aaccactgac ccgatgtcct tgctatcttg gacattgatg    720 

acttgcactc tttttccttg acacttatta taaaatggct tgatttaaaa cctaaccctt    780 

aatttttttt tcattattta gttgtacata cattggaagt tgatgcatct agtaaacata    840 

cctaaaaaga actgctgctc taagagttct cattttgctt tcagctgtac tctatttgag    900 

gaaatgactt aatttatgta tttttcttgt aattgtaatc ataaagcccc aatgaaaaag    960 

atgcaaattt gtgacaattt gttgaggtca atgtgatcta agttgtcaat atgaattatc   1020 

tgtttgaaaa cttgaatcta atttaaattc agaaatgtct aaatgtgttt agttaatgat   1080 

caaataaaca agctttaag                                                1099 

 
           
             2  
             938  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 2  
             
           
            2 

gaccgccgta gtggtgtcca cttcgattcc tggaaacctc gaggaaaaaa gtgaaataag     60 

caacggaaag atcgggaatc ctgaggcttg catcaaacat cccttacaga acaggtggac    120 

tctttggttt ttcaagaatg acaaaagcaa aacctggcag gccaaccttc gtctcatctc    180 

tacgtttgac acagttgagg atttctgggc gttatacaat catattcaac tgtcaagtaa    240 

cttgatgtct ggctgtgact actctctgtt taaggatggg attgaaccca tgtgggagga    300 

tgaaaggaat cggcgtgggg gccgctggct catcaccctg tccaaacatc aacggaaaat    360 

ggatctggac cggttctggt tggagacgct tttgtgtctc gtgggcgaag cctttgatga    420 

tcacagcgat gatgtttgtg gagccgtcat caacatccga gccaaaggag ataaaatagc    480 

aatatggacc acaaactgtg aaaacaaaga ggccatcaca cagattggtc gagtttataa    540 

ggagaggtta ggcgttccac ccaaagtgat catcgggtac cagtcccacg cagacacggc    600 

tacaaagagc agctccacaa ccaagaacaa gtttgttgct taaaggtttt gggtttgtct    660 

tctttccttg ttttgttttt ttcaaagtaa ctgaaagttt attcaccaaa agccttttct    720 

aaaatggaac aagtcttgcc tttactttgt cagaacattt gtttgtggtg agaatacaat    780 

aaaaaaatag taggtgtaaa atcctaatga ttgacttcat catgggatag aaatgtaagc    840 

agtttattgg aggtgattca tttgtgttcc aacaattctc tcagttttta cctttttaag    900 

ttgtttatgg agaattgctt aataaaggta catgaatt                            938 

 
           
             3  
             2862  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 3  
             
           
            3 

gctgcttcat cttcttggcc ttggtgtaaa agtcaaagcc aagttctggt ggttgagttc     60 

tttccagttc ctttttccaa tgccttcaca ctgcaacatg cggaagcctg agaaaatgtt    120 

tttgttattg aacctgatgg ctgcagtttc tgtcttgagt caagcatggc caaatgtgaa    180 

gcatggtctg cagtcaacca atggagttag atcggactgt gcaggtaatc tgatgaggct    240 

ttcattggat gaggctcttg cagttgggaa tcaacttcaa gtggaagcaa tcaatggcac    300 

acaacgtatt ttggtgacac ccagtctggc tgcccagtgt ggatacagca tggagtctga    360 

cccatgggga aacaccagga tctacatgtc tctgttgggt tgctttatgg ttagcaagga    420 

tgagtccacc ttcagcacca gcttgaaact gcacatgtac aagcacagtc cctctgatgt    480 

ggtcagccat gatgtgactc agacctgcag ctattctcgc tgggccttca gagatgtcct    540 

ctgtgacagg aactacatgg aagtgtcagc tcacatcgct cccagtcaac aaacaaaagg    600 

acaagttcag aacaaggaca actctcaaac aaataagctt cctggtgact ccaatgacgc    660 

ccctggaatc tggaagatga ccttttacac tcctgaacct gttgcgatgg tcctgaaaga    720 

agccgaacaa gctggttatg gtgcaacaat gagacaaact cgcctggcaa tcagaagccc    780 

ctatcatact tcagagactt attctgaaga tgttgctgga gtccccatgg aaatcctcaa    840 

agtgagcgtt taccacaaaa ctcaagaggg actgaatgtt gtcaacttgg cagctgcttg    900 

ccccacaggt ggcattctct tcactgacga gttcatctcc tggcacatcc ctcgccgcgt    960 

aactcctcta actgagggca aggtcaagat tgtagagctg tacatgggaa tcaatggaca   1020 

gaggctggaa aaggctcaaa tagctgcaag aggttactcc ctctcaacca cagagttcca   1080 

cattgttgtt gacatcccag tgggatcacc tgacggctac tacaagagcc atgctccaga   1140 

cttcctgtac cacatcacct acaccattga gccaatgctt gaagttctat ggaggccaga   1200 

gaatccccaa gaagaaacaa aatacaaaat tctgtttccc atcacaactc caccaatcct   1260 

tcatccaatc aacacctttg accgcacaat cccagaagag agggtgttta atgtccatgt   1320 

gggggctttc cttcatgatg tggtgctgaa gaacatcaca ttttctactg gagtccttac   1380 

tgttgaagag tgcaatgcca aagggtttgc agtccaggaa catcttctct ccaacggcac   1440 

aaaagtgttc tctattcaag tggcctttga caccgatccc atcctgaagc ataatcctga   1500 

gcccttggtt acaacctact tcctccctct gatctttggt tttgctgttc tgcctgagga   1560 

tctccctttt gcccacaaag tagagttgca ggcatctctg caggatgttg ttctgccaac   1620 

actcactgga acctgtgacc aggagcactt cttcgtttcc gtcaagtacg gcagtcaagg   1680 

gcatcacttc aagaccatgg tcggccatca ggacctgaca cctgaactgg gagagacact   1740 

agcctatcag gacaatggca cacacttcag cattgtggtg ccttatgctg cccccgtcac   1800 

tgcatttgag ctgatcacaa caaactcagt cagaaccagg ctgaatatgc tgctgtggga   1860 

ctcaatcaat caatgggttc ttggagacct ttacttgtct tgcagtttcc ctctggcaac   1920 

aacaaggtgc tactcaaatg gaacaatcag tgccatcgct gtcaaagtag agtctgtgcc   1980 

aaacctcagc ccaagctggc tgaccttgaa ggaccagtca tgcacaccag cattcattga   2040 

tgaccgcttt gctcactttg tcttccatgc tgactcgtgt ggaaccacta gactgttctt   2100 

cgacaactac atgatgtacg aaaatgaaat caggctgaac ttcaacagga aaggagttgc   2160 

ttacacatca ccagttgatc ctgattacaa gcaaaccatc tcctgctact atgtggtcaa   2220 

tgatacccag agaatctcct ttgcttctca accaagactc catgaaccca aagcagagat   2280 

tggattcgga cacctggtgg ttcaaatgag actagctcag gatgcttcat atcaaatctt   2340 

ctaccaaact gaagactatc cagttcagaa gttcctgagg gagcctctct actttgaagt   2400 

ggagctgatg cagtcaagag accctaaact ggaactggtg ctggaaaact gttgggcaac   2460 

aagcaaagaa gagaggaact ctctcccaag ctgggacatc attatcaatg gctgtgaaaa   2520 

cccagatgac agttatgctg cagtatttca ccctgtaatg aaggacagca gagtttcaat   2580 

cccatctcat gtcaagcgct tctccatcat gatgttcgcc ttcatccaga atgaccaagt   2640 

tctgaaggat gagattcatg tccactgcaa tgtagtgatc tgtgatgcca acacacctgc   2700 

agaaggcatc tgcaaaggcc aatgtggtta ctcatctggc attaaggcac accaaagcac   2760 

aaaaaaggga caaagaccac aaagctcaac caaccacaag cagatctcct ctgggcgtat   2820 

tctgttgaat aactgaatgt ccaaataaaa atttcttaaa tg                      2862 

 
           
             4  
             1278  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 4  
             
           
            4 

agtttgtagt cactaacacc ataatggggt ttggcaagtg gttgtttggt gttgtgattt     60 

tggcttgtgg tgcgtcagca caaaaccact ggacaccaca gaaacaccag cccctgtttc    120 

ctcatcaagc gcagccactg gtcactactt ttgataagtg caatgtggag gagggtgaca    180 

agattgaatg tgggacttcg gatatcactg tggagcagtg tgaaaaaatc aactgctgct    240 

ttgatgggtg gaagtgtcac tatggaaaag gagtgactgt gcagtgtacc agggatggtc    300 

agtttgtggt ggttgttgcc aaagacacca ctgtgcctcc cattgatgtg aactcgatca    360 

gtctgctgga gtccaatggt gacttctgtg gtccagttga cagcacctca gcctttgcca    420 

tttttcagtt tcctgtgact gcatgtggta ctacactcaa ggacgatgaa aactacattg    480 

tctatgaaaa ccacatgtct tcatcatatg aagtaggagt tggacccaga ggatcaatca    540 

caagagacag tcattttgag ttgttgttca tgtgcaagta ctctggctca gctgtggaag    600 

ctcttatctt ggaggtcaac cctgttcctg ctcctcaacc agttgcagct ctgggacccc    660 

tgagagtgga gctcagactg gcaaatggac aatgtctcgc aaagggttgc gtagaagaag    720 

aggaagcata caactcattc tacaacccag ctgaatatcc ggtaaccaaa gtgttgaggc    780 

aaccagttta tgttgaggtc agggtgcttg gaaggtctga cccaaacatt gtcctaaacc    840 

ttgatcactg ttgggccact gcaaccccaa atccccaaag tgttccccag tgggacctcc    900 

ttgttgatgg gtgcccttac caggatgaca gatactcaac gatattggtt ccagtggata    960 

gctcttctgg ccttgagtac ccaactcatt acaaacggtt catcagcaag atgtttgcat   1020 

ttgtggttcc agaaatatat actccccagg agacggtgta catccactgt gccacagttg   1080 

tgtgctaccc aagtagcaca aactcctgtg aacaacgctg tcatcgtcag cgaagagctg   1140 

cggtaaagat tccttcaagt cagaaggctc tggtctccag tggtaaagtg atcctgatca   1200 

agagtcctgc atctcacttg aaaagttaaa attgctcttg gccatctgtt ttaataaagc   1260 

ttgtcaaagc tcatgctc                                                 1278 

 
           
             5  
             1003  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 5  
             
           
            5 

ctgggatttg gcaatgtggt tctgtttggg ggctgttatg gcagtgaatg cagagctgag     60 

gacggactgc aggcctgact acatgtcact agtttggact gacagccggt tgcaggctga    120 

tccttccctg tttcgtcttg gtagctgctt tcctgcgact atcagccccc gggaggtggc    180 

ttttagcgtg acatatgatg actgtaactt caggagactt gtaactggaa atgagcttaa    240 

ctacaccaat gacctcatct acacatcttc tcctgactcg tatgtcaacc cttttagtct    300 

cccggttgtc tgctcatttg agaggcccaa ggactggtat cctatgactt atgacccaga    360 

gttttccaca tatggtgtag aagacttggt gtttcaagtt ggactaatga atgctgactt    420 

cacaggaccc tctgagtcaa atgtgtaccc cctgggctct atgatttctg tcatggctgc    480 

tgtggaccag caagaccatc agcctctatt gctgtttctt gaagagtgta tagcctccac    540 

cacacctgac cttcaccctg gagccgactt gtatccaata atcacaaata aaggatgcct    600 

ggtggatagt aaagtttcgc gttcaaagtt tgaaccaagg gagaaactgt ctgagattca    660 

cttgtctgtg caagccttta aatttgcttt gggacatgag gtgttcatcc actgcaccct    720 

ggttgcatgg gatccaaatg gactagatga caccaagaag gcctgccact acaacaaaga    780 

tcatggctgg gagttgctgg acaaccctgc atataatggc ctttgtgact gctgtgaatc    840 

cacctgcaag tcaagaaaaa gaaggaatct gtctgaaaag catggcttgg agaaaaaggc    900 

agttgttgga ccactcacaa taacggccta aagtcctgaa ccagcttgtg atttatttta    960 

agctgtaaat ccttgaaatg acattaaata aaatttgaaa atg                     1003 

 
           
             6  
             1110  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 6  
             
           
            6 

tggggacttg catttacctt ggagctgtga tccttgctgt ttttattgca gctggtgctg     60 

agccagatat taaagttgtc tgtgcaagag actctgtgaa agttaaatgg agggtttctt    120 

ctccctttgt gccgtatgct gctcgtcttt tccttggaag ttgcatgcca tccaagtggc    180 

aacttctacc ctctggtgat ggggaggcat tttttgatta caagttctct gaatgcaaat    240 

ttactaaaca gataaaagga aaaaatgtag tttataggaa tgaactcagc ttcaggccac    300 

aagcaaaggg aaggcccgtt gtttttaagc agccaattga atgtgtctat aagaggcctg    360 

agggttggat tcctccattc ttgaatcctg gatctggtgt gtctgagggt cacagtaatc    420 

tggtttttca catggcgctg ctcaatgaac aactaaccgg tgtggcaaag acaaatgtga    480 

tccccttggg ttcattcatg cctatatggg cagcagtgga gcagaagtcc catcagccac    540 

ttctgctgct catggatgaa tgtgttgcag ccaccacacc aaaactggat cctggcagcc    600 

aggttcacca aattgttgga aatcacggtt gtctccttga aagcaaacga gggagtgcag    660 

tgttccttcc acggtaccac tcatctgcca ttatccttta cattcaggcc ttcaactttg    720 

gacttggtga tgaggtctac atccactgta atctggttgt atgggatgag ggtgctcttg    780 

gccagagtag aaaagcttgc catgtaaaag accatggaag ttgggaactg cttgatgatc    840 

catcacgaag ctctgtctgt agctgctgtg actcagtctg cagttcaaag gccaaaagat    900 

cagtcggcga gtctggtaca tcaggctaca acacagtgtt gggaccattg gtgataaaag    960 

acctgtctgc tgcatctaat gctacactag ttggatctct ccctggaaga agtctgtagc   1020 

ggctgaacac gtgaaggaaa agtctctgtc ggagtgaaat ttcagctgta gtctgtatgc   1080 

tttaatccaa ataaagtttc tttaaatctt                                    1110 

 
           
             7  
             1258  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 7  
             
           
            7 

cagttttctt ttaaaccata ttttatcctt acttccatac tggctttctt gtggtgtccc     60 

taacacctcc taaggctcgt catgaagaag tttttggctc attcagcctc cctcgtgctg    120 

ctcatttctt ttctccgtgg agttgcaatt gccatccgaa ctctaaaaga aggtcctatg    180 

attgatgcag acggaaggga atataaaact gcttctttga cagaagactc cagcccgaga    240 

gcaagcgata gtgtccgtgt ggagtgcaca gaagtgtcca tgattgtcta catacaagca    300 

gacttctaca gaactggacg ccttgtgtct ccaggggact tgtttttggg aggtgcagag    360 

cataagcagg acagtcggtg tcgggctgtt gtttctggac acaatgagta tgtcattgaa    420 

gctggcttgc aagactgtgg ctccaagttg actataacgg atgatgatgt gatctactca    480 

aacaagctgg ttttctcacc agtttccaat caccacacta ttacaaggat gactgatgct    540 

atagtccctg tgtcctgcca ttacaaaaga acacacactg taagcagcag taatactgaa    600 

caggcgccca tgacgttctc tcagtcagca aagttctcaa ccaagaactc tgctttctct    660 

ttgaagctga tggctgatga ctggttaacg gagatgttgt ccagcaagtt tcatcttgga    720 

gactttctgc gctttgaggc aaagtatacg ggcccagagc ccaggcagct ttttgtcgaa    780 

agctgcgttg ccactctgac acctgacgca acgtctgtac ccagatacta cttcattgaa    840 

aaccacgggt gcttcactga ttcaaaagcg gggtcagttg cctcattcct acccagatcg    900 

agagccaatt tgcttcagtt ccagattgat gcctttctgt ttcgtaatga tttgagaaac    960 

actatctaca tcacttgtaa cctgaaggct accctccaaa tgaggaccac cttgactgac   1020 

aaggcctgca attatgtgca ttcaagctgg aaaagtgtgg atgagaacga tagtgtttgt   1080 

tggtgttgtg acagtatttg ctaccaaagt cttcccagag atgatgatct ttgtgacatt   1140 

gtcactcttg gtccactgag gattatctct aacaagtaaa gagcaagtga caaactgtct   1200 

gccagaggct cttggattaa acctgcgcaa cggtcttgta tgtccaaaaa taaagttt     1258 

 
           
             8  
             1509  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 8  
             
           
            8 

gcctcgtcgc tccttccaaa ctctgaggat ggactgcaac ctgcggctgg ccgtttcttg     60 

ctggatcatg gtcttttctt gggtttcccc tcttacagag agtcgccaga cgaacagccg    120 

aggttctaca gaaaggcact tccaacctcc agtcgggacg cacggcggtc tgcagcctcg    180 

gctctactcg gtgaagcagc agccggcccc ggaggtaccc gaacagcacc gccccgtcac    240 

ggtcatatgc cacccggatt ccatggaggt tgtggtgaag gccgacatgt ttgaaacggg    300 

cctgaatgtg gacggtggac atctgcgact gggttccaac actctgggcg cgggcggtga    360 

gtgcggggcg gtccagaaag gagaggacga attcaccatc tgggccctgt tgtccgactg    420 

cggaaccaaa ctctcatcaa cagaagagaa gatcatttat tccaacgttc tgatctactc    480 

acccgaacct tctgctgatg ggttgttaag attggaagct gcaactattc cagttgaatg    540 

tcattatgac aggagatact ctgttgatgg catttccctt gaatcaactt gggttccctc    600 

tgtctccaca acttctgtga acgaccagat agatttcaat ctgaaactca tgactggtga    660 

ctggcagtct gagagggagt cttacacata tttcctggct gatcccatta attttgaagt    720 

ctctgccata gtggaaaatc acgtccctct gcgggtgtat gtggaccact gtgttgctac    780 

ggcaactcct gatgcagagg ctaatttaag atatgaattt attgaacata agggctgcct    840 

cgttgatgct taccttacaa actccggcgc acgtttccta ccaagaactg aggaacataa    900 

actgaggttt cagctggaag ccttcaggtt ctatcaagaa cccagcaacc agatctacat    960 

tacttgtgct gtgaaggctg ttcctgctgt acaggccgtc agttctcaga accgagcctg   1020 

ctcctttatt gagaacagat ggcaatccat agacggtggt gatcaggtgt gcagaagctg   1080 

tgacgtgttc aggcggggtc aggaaccgca agctgtgcca tctcctaaaa tgccagtgaa   1140 

cgccaaagac caaatcggtc tttcacagaa aaatatagtc cacaataaag ccgagcatca   1200 

accggcttct tacgtccatt tttggccggg agcttatcag agccatcact ccaaacctca   1260 

gcagtccaac agatttatga agagggatgc tgacaacaaa tttcatcaag ctgtccaact   1320 

ggggcccctc gttgtgctac cgtcaagaaa agtagtttca gtggcaacaa atttttctac   1380 

atggtcagaa aagaacaaca cctcctgaga cctggaacac ctgatggagg agtctgagtt   1440 

cgtttgagag atttggtctt gttgggctaa agaattttgc tgcaatccaa taaattaagt   1500 

ttctaatgt                                                           1509 

 
           
             9  
             1929  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 9  
             
           
            9 

cgcggccgct gggtcagcga ggagcacttg cagtctgatt tttgggtgct gctgcttcag     60 

tcattttgat ggctccctca cggttaaaga ttagctgcct ttttgggcat ttgactgcct    120 

tctgtcttca gttgacactt gcgtttccac cattgcttta cgtgccgcct gtttctctca    180 

aaagccaaag ctccttgccg agtgtggtcc agcagccaga ggagccggtg cccgttaaca    240 

cagttggtgt gctctgccac cctgattcca tggagctcag catcaatgct gacctgtttg    300 

aagtgggagc gcctgttgac gtccgtgagc tgcgtctcgg agtcgagcac agcgactact    360 

gcagtgcaac agcatcatca gactctgagt acagaatcct cgtgggcctg gaggactgtg    420 

gcaccaaaca ctggatgaca gaagactctc tggtctacac aaacctcctg atttatactc    480 

cccttcctgc acttaatgga ataactcgaa tggaggaggc cgttattcca attgaatgcc    540 

aatataaaag gaagtacagt ttatccagct catcactcgt gccaacttgg gttcccttca    600 

cgtccacaca agctgctgtg gaaactctcc agttcaacct gaggctcatg accaatgact    660 

gtgccataac tgtaaagaaa cctcagaggc caaagaaatc ctggcagaaa cgtctgaatg    720 

gacacaacca gccctttgtg gaggaaaacc tgcagcggct cttcgctctg cgcatgcgaa    780 

tctccagacg tgccaaagtg gaaaccaacc ttgccggtct cttcaacgag cgcaagatcc    840 

ctcatttcgt tgacccagag gtcaacctgc gtaacctgtt tggcatcaaa cctcccattc    900 

ctttggaaag tcctgctgtg gcaccagtta aatgaaaatt cagatctgat gagttatttt    960 

aggaagttgg gtctttttag tttaaattgg gcatttatgt tcaaagaatg ttaaaatttg   1020 

gctgcacgaa aggggtgcca acacgttctt cctcggcgag ccaatcaaca ttgaggcatc   1080 

agtcagggtg gatcatcaca tggggctccg gctgttcctg aacagctgcg tggccacact   1140 

tgaacccaac atcaaatctg atcccaaata tgtctttatt gagaatgggt gcctgctgga   1200 

ctcccagctt ccaggctcaa agggtcactt cctgccaaga acaaaagaca atatactgca   1260 

gatgactatc gattctttta aattccataa tgacgaacga ggacagcttt acatcacatg   1320 

tcatctcaat gcggtgccag tggatgatgc agaagcacaa agcaaggcct gcacatatgt   1380 

aaatggaagg tggcggtcag ctgatggaaa cgactacctg tgtggattct gtcaaagccc   1440 

caatgaagcc agtaaagcac tcagcatccc tggagtcttt aatcctcgta gctttggaaa   1500 

atctgcagac atggagccca tgtggagaag tggactcaag actaataagg tttgggaaca   1560 

tgaagccaga ctggggccag tgactattct gccttcatgg aagagtgggg ctcttgctgc   1620 

acatgaacta cctccagttc tccataagat ccacaaaacg gcgctgtacg gcagccactg   1680 

gaggagcggc ctaaacacaa tcgagaagag cctggttcca gaaccatcaa gttcagaact   1740 

agaagaggat gacagtgatg actatgatga gtctgactct gacctggcgt ttgtgatgaa   1800 

gtccttggag attgcccata ccaacactac cttccccatc gtcacatatg acctggagcc   1860 

ctctaagatg aatgcaactg ctgctaaccc tgacctttct gataaacatg acccaaagaa   1920 

ataaaatgc                                                           1929 

 
           
             10  
             919  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 10  
             
           
            10 

gaagattaga tgatgaaagc tgagaaagct gttggggctg ggccccagca ggtgttcacc     60 

tgcacccacg ccggatgtgg agcctgcttc ccgagggagt ggaaactaaa ggcgcacgaa    120 

actgtgcaca ccggagagcg tccttgcgcg tgcccaacag ccgggtgtgg tagtctcttc    180 

aagagaacat cccatctgaa aagacacgtg ctccagcata aaggagtcaa agggttccaa    240 

tgcaagtttg caaattgcgc aaagagtttc atcgacgctc aaaggctgaa gaagcaccag    300 

aacagcgctc atgggaatca caaattcaag tgtaatcaac ccaagtgctc cttgagcttc    360 

aagaagcgca gattgttgaa gctgcattta aaggagcata atgtccatcc gaacttcaaa    420 

tgttctaaca ttaggtgtac tgcaacgttt gactcccata ttgcacgcaa agcccatgag    480 

aagaagcatg caggttatag ttgccctcac aaagactgcc aggttgttga acacacctgg    540 

agcaaacttc agaggcacct ggccaaacac ccagtctcat ttacctgtgg cgtgtgcgag    600 

aaggtgtacg acaaagcagg tgctctgcgg cggcacaaac ggatccacgc ttcccataaa    660 

cctgtgctgc tgtgtccaag agctaactgc caggcctact tcaccacgac ctttaaccta    720 

gagcatcaca ttcgcaaagt gcatctccag ctcctgaagt ataaatgttt cttccccgac    780 

tgcccacgca catttgttat gcgggagagc atgcatcgac acatggttca tcatgatcca    840 

aaatttcctt taaagtaaaa ggctaaataa aaatgctctg tagtgttatt gacccaataa    900 

aatgagtttt gttttgctc                                                 919 

 
           
             11  
             1557  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 11  
             
           
            11 

ggaaagcttc agttggtaag acgtcagtgg ctcacattca cctacaacca tggcgaagga     60 

gaaggttcac gtcaacgtgg tcgtcatcgg tcacgttgac agcggtaaat ccaccaccac    120 

tggacacctg gtctacaagt gtggaggcat tgatcccaga aagctggaga aatttgagaa    180 

ggctgcagct cagttgggaa agagttcctt caagtttgcc tgggtgctgg ataagctcaa    240 

agctgagagg gagcgaggga tcaccattga tatctcactt ttgaaattta acactcaaaa    300 

gtacaccatg actataattg atgctccagg gcacagggac ttcatcaaga acatgataac    360 

tgggacgtcg caggcagatg tggccctcct gatggtctcg gcagccaaag gggagtatga    420 

agccggcgtt tccaggagcg gtcagaccag agagcacgcc ctgctggcct acacgctggg    480 

ggttaagcaa atcatcgtct gcgtgaacaa gatggatctg accgagcccc cttacagcca    540 

gaaacgctat gaggaagtga tgcgcggcgt gagcggcttc ctgaggaaga tcggctacga    600 

cacgaatgcc gtgcccttcg ttccagtttc tggatggact ggggagaaca tgatcagcgt    660 

aacccagaag atgccctggt accaaggctg gaaaatcagg cggagggaag ggccttcaac    720 

tgggaagact cttcttgaag ttctggactc tattcagcct ccggtgcgaa caatcaacaa    780 

acctctacgg ctacctctgc aagacgtcta caaaattgga ggagttggga ctgtgccagt    840 

ggggaagatt gaaacaggcg tcctcaaacc cggcatgacc ttggtgttct ctccagctaa    900 

gctcaccgca gaggtcaagt caattgagat gcaccaccag ggcctgcaga cggctctgcc    960 

ggggcacaac gtcgggttca acatcaaaaa cgtgtcagtc aagaacctgc gccgtgggga   1020 

cgtggccggc aacgcccaac aggatccacc ttcagatgtc cgcagctttg aagcacaggt   1080 

gattatcctg aaccatcctg ggaagatcaa ggcgggatac tctcctgtcc ttgactgcca   1140 

cacgacacat gtcacatgcc gcttcaccga gctgaaggag aagctggacc ggcgcaccgg   1200 

caagaagctg gaggagcagc cacaaaccct ggtgtctgga gatgctgcca ctgtcaaact   1260 

ggttcccgtg aagcccatgt gtgtggagag cttcttcaca taccctcctt taggccgctt   1320 

tgcggcaaga gatctgaagc agacagttgc tgtaggagtc atcaagtcgg tggagaaaga   1380 

ccaggggtct aaagctcaaa agcttcaagt ttgtaaataa gtgctctgta aatgtatgct   1440 

gcgaaaaagc tttttatttt ccagtgaatt tcagtttgtt taaaatgtat aatttgacca   1500 

gaaaagatgc agctggtata tttctgttaa tttaaacaaa taaaaactgg tttggag      1557 

 
           
             12  
             865  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 12  
             
           
            12 

tcattgttca cagttgacgg gctgctttaa gatggcaaag aaggtgctga tcgtgtatgc     60 

ccaccagagc tctagttcat tcaattctgc agcaaaaact actgctgtgg aagttttgac    120 

cactctgggc tgctctgtgg aagtttctga cctgtatgct atgaatttta aagcaactgc    180 

cactgctgag gacattaaag gtgaccttaa ggatgctgaa aattttagct acttggatga    240 

aagtaagctg gcatgggagg aagggagact aatggatgac atcactaagg agcagtctaa    300 

ggtcattgag gccgacttca tcatctttca gtttccaatg tactggttca gtgttcctgc    360 

catcatgaag ggctggattg accgtgtgct cacaaacggc tttgccttca cacaagagaa    420 

acgttacagc cagggaatct tcaaggaaaa gagagccatg ctgtccttca ccactgggtc    480 

acttgaatca atgttcagtg ctactggcat taatggagac atgaatgtca cgctgtggcc    540 

gctgcagaat ggaatcctgc actactgtgg cttccaggtt ctggcccctc aaatcttctg    600 

ggctccattc tcagcaaccc ctgaagctcg tagctgcatg ttggagggct ggcgtgcacg    660 

actgcaaggc cttctagagg agcagcctct gtcattcatt tccctggact gctttgacaa    720 

aaaggggttc caattgaaat ctgatgtcca ggagaagcac gcagccaagg actttggtct    780 

ggcggtggga atccacatgg gaaagcctct gccacctcac aaccagatga aagctggatc    840 

ttaaacatgt ttgcttgaat gatga                                          865 

 
           
             13  
             705  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 13  
             
           
            13 

cttttgggtt ctagtttagt ggtaactgta gcagaatgag gtttctgtgg atttcttgtc     60 

ttctcattgg aagcatctcc tgtcttcctc aaggaggata tgatccatcc ctgtttctat    120 

acactggaca agctccatcc tatgagaaac cttctgcaca gtccagtggc tacagcagtc    180 

cacaaggcta ttacagtgct ggcaccaaca ctgctggagg ctctacagac agtactgctc    240 

ccatgtggta ctctgcttcc tatcctgaac aagagccagc caagccaact tatcagagac    300 

ctgcacagtc cagtggttat ggcagctact ctggttctgg atctcaacag tctggttccc    360 

agggcgctca gtctggagtt ccaggcagcc agcaccaggt tgaacaggag agctggagct    420 

cctcatctga cgacgaggaa gagcccgagt tcactccagt gagtgaggag gatcaagtgt    480 

acgctttcaa gtctcgctct cgctacaacc agaaacggct gctgttcagt cagttccgct    540 

acaccccaac agaaccccgt gttcctcaag aaccagtgtt cccgtacccc ggcaagtctc    600 

atcagggcaa aggttcagct aaaggacgcc gctaagatct ggcttttgtt ttgaggaaac    660 

cgactgattt attctgaaga ataaattaaa atcttaaaat gttac                    705 

 
           
             14  
             725  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 14  
             
           
            14 

ctgaggtttc tgttctttgg gggtggagca gctgcagaaa aatgaggttt ctgtggattt     60 

cttgtctgct cattggaagc atctcctgtc ttcctcaagg aggatacgat ccatccatgt    120 

ttctatacac tggacaagct ccatcctatg agaaaccttc tgcacagtcc agtggctaca    180 

gcagtccaca aggctattac agtgctggca ccaacactgc tggaggctct acggacaata    240 

ttgctcccat gtggtactct gcttcctatc ctgaacaaga gccagccaag ccaacttatc    300 

agagacctgc acagtccagt ggttatggca gctactctca acaatctggt tcccagggtg    360 

ctcagtctgg agttccaggc agccagcacc aggttgaaca ggagagctgg agctcctcat    420 

ctgacgacga ggaagagcca gagttcactc cagtgagtga ggaggatcaa gtgtacgctt    480 

tcaagtctcg ctctcgctac aaccagaaac ggctgctgtt cagtcagttc cgctacaccc    540 

caacagaact gcgtgttcct caagaagcag tgttcccata ccccagcaag tctcatcagg    600 

gcaaaggttc agccaaagga agccgctaaa gatctgactc catgtgttct gtggctgttg    660 

gcaaatacct aaatgcaagt gctgtgtggt ctttaaaata aatatttaaa gttcgactgt    720 

cgtgg                                                                725 

 
           
             15  
             728  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 15  
             
           
            15 

ctgtctcttg tgtataatcc agcagcattc acatcatgat gaggctcttg ttactttcat     60 

gtttcctcct tggacgtatc acctgctatc ctcaacaagg tagtggtggt cattttctcc    120 

cataccaagg tcaggctcca tcctatgaaa aaccattgat gcagtctggc tacagtggct    180 

ttcctggtgt atacagcagc agcatgaaca cagctggagg cagtggaagt cctcccatgt    240 

ggtactctgc ttcctatcct gaacaagagc cagccaagcc aacttatcag agaccagcac    300 

agtccagtgg ttatggcagc tacggcagtg ttgacagcag ctactctggt tctggatctc    360 

aacagtctgg ttcccagggt gctcagtctg gagctccagg cagccagcac caggttgaac    420 

aggagagctg gagctcctca tctgacgacg aggaggagcc agagttcact ccagtgagcg    480 

aggaggatca agtgtacgct tccaagactc gctctcgcta caaccagaaa cggctgctgt    540 

tcagtcagtt ccgctacacc ccaacagaac cccgtgttcc tcaagaacca gtgttcccgt    600 

accccagcaa gtctcatcag ggcaaaggtt cagccaaagg aagccgctaa gatttgtgag    660 

ggccactgaa gatcacctga tttgactttt gtaatctaca gactccgcta ataaatgaat    720 

taaaattc                                                             728 

 
           
             16  
             729  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 16  
             
           
            16 

ggcacgagat aatccagcag cattcacatc atgatgaggc tcttgttact ttcatgtttc     60 

ctccttggaa gtatcacctg ctatcctcaa caaggtagtg gtggtcattt tctcccatac    120 

caaggtcagg ctccatccta tgaaaaacca ttgatgcagt ctggctacag tggctttcct    180 

ggtgtttaca gcagcagcat gaacacagct ggaggcagtg gaagtcctcc tatgtggtac    240 

tctgcttcct atcctgaaca agagccagcc aagccaactt atcagagacc agcacagtcc    300 

agtggttatg gcagctacag tggtgttgac agcagctact ctggttctgg atctcaacag    360 

tctggttccc agggggctca gtctggagct ccaggcagcc agcaccaggt tgaacaggag    420 

agctggcgct cctcatctga cgacgaggaa gagccagagt tcactccagt gagcgaggag    480 

gatcaagtgt acgcttccaa gtctcgctct cgctacaacc agaaacgact gctgttcagt    540 

cagttccgct acaccccaac agaaccccgt gttcctcaag aaccagtgtt cccatacccc    600 

agcaagtctc atcagggcaa agtttcagcc aaaggaagac gctaagatct gtgagggcca    660 

ctgaagatca cctgatttga cttttgtcac ctactgactc tgagctaata aatgaattaa    720 

aattccctc                                                            729 

 
           
             17  
             696  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 17  
             
           
            17 

ggcacgagag ggtgctgttg gtttgtctgc ttgttggaac tgttacctgt gttcctcaag     60 

gaggtggagc ttatcagccc agagggcatc ctccattctc tggacaactt cagtctgagc    120 

cagtctatga aaggccttcc ggacagtcgg gttatagtgg cgctccggga tattttacca    180 

gtggaaccta cactacagga ggcagtggaa gtcctcccat gtggtactct gcttcccatc    240 

ctgaacaaga gccagccaag ccaacttatc agagaccagc acagtccagt ggttatggca    300 

gctacggcag tgttgacagc agctactctg gttctggatc tcaacagtct ggttcccagg    360 

gggctcagtc tggagctcca ggcagccagc accaggttga acaggagagc tggagctcct    420 

catctaacga cgaggacgag ccagagttca ctccagtgag cgaggaggat caagtgtacg    480 

cttccaagac tcgctctcgc tacaaccaga aacggctgct gttcagtcag ttccgctaca    540 

ccccaacaga accccgtgtt cctcaagaac cagtgttccc ataccccagc aagtctcatc    600 

agggcaaagg ttcagccaaa ggaagccgct aggatctcgt tgagcgtcac tcatgatgtt    660 

ttgatgttga gctgctgaaa agaataaaaa aaatac                              696 

 
           
             18  
             731  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 18  
             
           
            18 

cttgggcatc agtcaatagc aaccagcaga atgagggtgt tgtggatttg tctcctgatg     60 

attggaagca tcaactgcct tccccaagga agtgtcccaa atatggcagt ccctcgctcc    120 

atgtggcttc ctccttacta tgggcaagaa ccatctagac catcctatga agagccttct    180 

ggacagtatg gtggttatcc caccttccca ggatcttaca gcccggagcc tcaaactggg    240 

ggcagtggaa gtcctcccat gtggtactct gcttcctatc ctgaacaaga gccagccaag    300 

ccaacttatc agagaccagc acagtccagt ggtcacagca gctacggtgg tgttgacagc    360 

agctactctg gttctggatc tcaacactct ggttcccagg gcgctcagtc tggagctcca    420 

ggcagccagc accaggttga acaggagagc tggagctcct catctgacga cgaggacgag    480 

ccagagttca ctccagtgag cgaggaggat caagtgtacg cttccaagac tcgctctcgc    540 

tacaaccaga aacggctgct gttcagtcag ttccgctaca ccccaacaga accccgtgtt    600 

cctcaagaac cagtgttccc ataccccagc aagtcacatc agggcaaagg ttcagccaaa    660 

ggaagccgct aagatctgtg gtctcctggg ttactgatat tttcaaatgt gaaattaaag    720 

tttcctctga c                                                         731 

 
           
             19  
             761  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 19  
             
           
            19 

tgttttgttt tgggtattag tcagtattat ccagcagaat gagcagggtg ttgtggattt     60 

gtctcctgat gattggaagc atcaactgcc ttccccaagg aggtgtccca aatatggcag    120 

tccctcgctc catgtggctt cctccttact atgggcaaga accatctaga ccatcctatg    180 

aagagccttc tggacagtac ggtggttatc ccaccttccc aggatcttac agcccggagc    240 

ctcaaactgg gggcagtgga agtcctccca tgtggtactc tgcttcctat cctgaacaag    300 

agccagccaa gccaacttat cagagaccag cacagtccag tggtcacggc agctatggtg    360 

gtgttgacag cagctactct ggttctggat ctcaacactc tggttcccag ggcgctcagt    420 

ctggagctcc aggcagccag caccaggttg aacaggagag ctggagctcc tcatctgacg    480 

acgaggacga gccagagttc actccagtga gcgaggagga tcaagtgtac gctttcaaga    540 

ctcgctctcg ctacaaccag aaacggctgc tgttcagtca gttccgctac accccaacag    600 

aaccccgtgt tcctcaagaa ccagtgttcc cgtaccccag caagtctcat cagggcaaaa    660 

gttcagccaa gggcagccgc taggatctgt catttcagga tcattaatcc atgactgctg    720 

tgcaggtttt catgtaccag tctaataaaa taccattcct g                        761 

 
           
             20  
             629  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 20  
             
           
            20 

tacgactgca gtgaaacttt tctagtttaa tttagcaggt tgctgctttt caaagaccaa     60 

cgtgatgctt cagtctgtta tgaggttgtt ctatatcagc cttttcctct tatactttgg    120 

agcctgtgtt ccacttaaaa aaagtgaaac ttctctcggc tctggcttca gttattccag    180 

tcctgggttt ggctctgact actcgggacg cggttcttcc atttttggtt atgactctcg    240 

tggcgatttt ggttccggct cgccgagaaa gcaggctgca ggctttgatc gtttcattgc    300 

agaaatcttg agcctgagac cttctcgttc tttcccacgc cgtgcctgga cctccaacca    360 

ggtgcctctc ggcatggttg agccacgccc cgtgtaccct tcatcccacg ttgtcagaac    420 

gagcaatggc taccagcgag ctcgggactt ccggagtgat gccaagtacg ctcaagatat    480 

ttttgaccac atagatgagg acggtcaaca agagggccac caaccgactg gaccaacggg    540 

tcaaaagacc tactgaggta aaggtgaaga atctactacc tggacagaca gacatacata    600 

caaacaaaca aataaatgtt aatctgtct                                      629 

 
           
             21  
             843  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               cDNA for gene 21  
             
           
            21 

gtttaaggag ggcagtgaat tcccaaagct tgtgttggag ctgcaggcag gagccatggc     60 

tgctgggttc ctgattagca ggtttctgct gatttttgtg ttgagtgaac taaagtactc    120 

atcggttttt ccatcagttg ggagttgggg gagttttcag gtaacgttcg accctggctt    180 

tcaccatcac cacaaaccca ctaatccatt gatgaattac tggttaaaac ttaaggagtt    240 

gccaaatctt tggcaccaca cacggaacaa accgctgtgt tgtgatggtg actcaaaggt    300 

gcctcgccat cctgtcaagc ctccagtccc aatctgtgaa ccaacaaacc agggaccaga    360 

ctgtcctgtg aaacacggcc caacccatcc tgaaccaaag tggcccatcg tcagtcatgg    420 

tccatcccat caccacttgc actggccttt ctttcgtgtc ctgcatggac ttctctgtca    480 

ccagcaccct tgtcctcatg cccacagcca cgcctacgat gatgaccgct gttctgcaca    540 

tcagcatcct cgccactgtg gaaaacacaa acaccaccat gggcctgtat tctaccaccc    600 

tcaccatggg cctggacatc atcatcatca tcatcaccac aaccaccagc agcaggtcaa    660 

ggtccccccg catggctgtg aacctcacag taaattatgt tcatgatgtc caagaagtga    720 

ttcaagcttt gttggctcct ctgagtcaaa gcagcctgac ctccacggag gactctactt    780 

ccaggttggg ccaggaatac tgagctgtgc aggtgtctgc atcaataaag atttctgata    840 

gag                                                                  843 

 
           
             22  
             13502  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               Genomic DNA for gene 1  
             
           
            22 

gatcttaaac ccgagcccga gtcagaaccc gacccgacgt ggggcactaa atgacaatca     60 

ttacgttcgg gtcggttcgg gccgggcttc tctctcgttg acttttttaa taaatatatt    120 

gtaattgctg caaagaagct ccttaggcgc gcgttttcaa acaactattt attaatcctg    180 

tatgtcagaa cggttaccgc acgaaccgaa aggcacagcc agcttcttct tactcagagg    240 

gagaacgttc accgaacgca cacacacaca cacacaggta gaccccgccc cctctatccc    300 

accagtcacc agcccgccgt tgaatgacac acactgtggt ccagcaactg gcagaaagag    360 

gggggcgccg gccacccgca gctctgcgca cgtgtcgtga atgtaatctt ccctccgcaa    420 

gcccaaatct atttttttaa ggtatccccg atatggagca gcaagaagtc aaggataaac    480 

ttaagacagg ggaactgaaa ggcaaacacg caccgcggct gcagaatttc tccgcgtaat    540 

gaactttcac tctctttgtc tggcctctaa ttcgagtaag ccctggctgc agaattgctc    600 

aacatagtgg aatgctggaa cgctgaggaa tgtgaacacg ctgcattcca ctatgttgtg    660 

caattctgca ggctgcagcc aggggtagtt gtcttattct gtgttccagc gttatttcgt    720 

tgtgcaaatg catttatcat gtagggagga atctatgcgc agcgcttccg gcggaactct    780 

atgccacaac agcgagcttg actacgcgcg cggctgcagc gaccactctc tgttcgaaag    840 

aaaataactt tcatgatcat aaaaatatgt agattattta acctttaaga aatctggcgt    900 

ttttttctgc caaaaatact atgggataaa ttaaactttc gggtttgctt cgagctcggg    960 

cctgcaaatc aagttaattg gtcgggttcg gaccgggttc ggctttaatg cccgtgggcc   1020 

tgtctcgggt cgggctggat tctttcggtc cgatcttacc tctaagctga actcacagtc   1080 

tttggttcag agggttaact gcaccacagg caaagcagca atgaggagag tggatgcaga   1140 

tttaatccaa aaaagagaaa agacaaagta agtttttaat taaataagga acagaaacta   1200 

aaattcataa cagaagctga ctagtaccta aacatcctca tgaagaatgt tttgatttat   1260 

tttggtaatg aaattgcttt tgtaactgaa gttgctgttt aagtgaactt aaaaaaccct   1320 

ggttatccta tctgcttata catttctaat gctgttaatt tattcctttc tttcatctat   1380 

tgttatcaca ctagttttca taattacttg aaacctttgt acttggcttt gctaaaaaaa   1440 

aaaaaaaaac atttcaggga attattcttt tttgaatctt caatacttca atacttcaat   1500 

gtttcaagca gtagcatttg tttttctttc ttgttgcatc aagtggcctc ttggcaaaaa   1560 

agtcaatttt ataccaacca aatgtcaatg cagggcaatg agctctgaaa tctgagtttt   1620 

agattgaagc aaaacaagaa atatgttcca gtttagaaac ctccaaggca acaaactcaa   1680 

gtgtcaacca caaagaaaaa gtataaggca gaaaaaaact gttctataaa tgtcttgaag   1740 

tggcaaattt gccacaacag cccccattat tctaatttat ttgtgtcctg acattcatgg   1800 

aaacaggttg tttacttcag cataaggatt actgcacaga aattgatgat acattcacac   1860 

aacataaagt ttgtgtcgaa tggcttttgt gtaaaggggt tgtttttttc ttgtgtttct   1920 

ttcaagtggg ccggtaaaac ataatgtaat tgcatactgt ttagataatc aagtgtaatt   1980 

atgccaggca tcctgacgtt cttttgagga gttcaactat acctggaaat tatggcggga   2040 

gaaaatgttc tttgtatatg tgtccctgac ctaaactgga gtgttctgca acacaatctc   2100 

aatcaaatca agagatgagg agaaagtttg ctgctagtct aaatctgcaa catatacaca   2160 

gtacatctga catactcaag ctagccagtt tttcaaagtc catcaaaatg tttaaatgtg   2220 

accctgcgtc attttcctga ttggtatgat tcagggtttt gagaagtctc aaatttaaaa   2280 

aatctttagt aaaatgcagt ttatttgtgt ttctctttgt gcctctgaaa gattcacatg   2340 

tacttaagtc acaggaaatg gtgctgcaaa aataagagta tttccccagc ctggtcacac   2400 

ttctgtggga tggcatcctg ctcttcaact acccttagcc cactaggcac ggacatatct   2460 

attggatatc taaattatgg ctattttaaa gctcacagct atagatatga attatgcatc   2520 

tgacagacat caattcatag aggtctattg catatccggg tttagatctc tagcaaacga   2580 

ctttcccgca gctaatcgtg tcacggtccc atcatgcttg tgcattgtaa acaagccgtt   2640 

tgggtaccat agtttcttcc cactggacac agaatgtcta gacatctatt gtagatgtct   2700 

actggacatc tcagacaact ctattttgtt acactcagat gcctactaat attgttctgc   2760 

atacagaggt ctcttatcta cctgtagaca tcaaaccttg gatatttttt agatgtctat   2820 

tacagaactt ttttttttaa tttatttact ggatataaaa ttgaaaaccc agactgaaca   2880 

tgtttctatc agtgagattg tggtgtgctg tcattctttt tatctttttg ttatcaaata   2940 

taaatataac aacattggtg tagagtccac tttttgtcct aatatataaa cacatctgtt   3000 

ttctaaacca gttttatcct ttacagggtc acatggctgc cagagcttaa cctgacaact   3060 

gatgggcgaa gccagggtac accctggaca tgttgccagt ctgttggcca aaaacagtct   3120 

gtgcagggcc acaatcacat acaaataggg gcaaattagc atgatcaatt caatcaagtc   3180 

tgtattagca tgtggttaac aaatcccaac agtggagcaa catctcaccc atgtggtatc   3240 

aaacagtctt ctttttactg cttagatttt aattattggt tttaaatttt gtttcattta   3300 

ttattggttt ttcatttttg tgtatgcagt tatatctgtt tgctcattag ttaaataggt   3360 

gtcttactta acttttgatt tctattccct ctcgatcatc ttccctagtc tggaaaagac   3420 

agacctgaaa gaggtaaagc ttatgtaatg tactttgagt tgcgaagtag ctctgtcttg   3480 

ggctaactag agtgtacatg taaatgtacc taataaagtg atttgtaagg tacgctcact   3540 

cagttcgtga acacactccc tcagttatct ttgcgcttgt tcaattaatg gcagtcatga   3600 

aacgccatag tcccaactga gatttgaacc ggaccttcac gctgtgaggc aatagcgctt   3660 

ttcagtagaa atgtttgcta cttcttgtaa tgcagaactt tgtgattgct agttgtctgc   3720 

catcaaaaag gctgtatggc ggtgtagagg ttagctcttt caacttaaag taagaagaag   3780 

aacctggttc aaatctctgt tggtaccttt ttgtgtgaag tttggacgtt tttcctctgc   3840 

agatgtggat tttctcctgc ttactcccac agttcattaa taggtgtcat agtataactg   3900 

gttcctgcaa attgctcctg tgtgttttta tggccctttg acaaactggt gacatgatta   3960 

aggtgtactc cacccacttc ctgggttgga accagcaaac tggtgactcc aaaagagatt   4020 

cagagggcct gaagatgaaa ggatggatgc aaaattgcag tggttatttt taaataataa   4080 

tcccttaatg gacatccatg tgcaggagac ctctatatgc agatatttct tagaaaacta   4140 

ttgttagaca tctaacagag tatctagaca tcctgtggct agcgggattt accagtcagc   4200 

cagcatgttg gtcactgtga aaatgtctga acaccatagt gttcggtgga gttgaggtca   4260 

gatctggtgc tacgtcattc tccttcactg cccaaattaa aaggtagtcc tttttataga   4320 

gtggttacat gtggttacaa atgaaatttt gctgggacat ataaatctgt gcttatgtgc   4380 

ccattcctag tgctccatgc aattgtggaa caaggggagt atcatcttga tattcttcat   4440 

tgttcaaata tgtgacgtgc aaaacctgag gtggaaaaac aacagtcaac aaggctgcat   4500 

gcgtgcttgt atttgcagat cgaatcatct taattctata gagacaaagt gctctggttg   4560 

gctgctttgt gatgattaaa aactgttaaa ttagcagcaa tcagtctgaa caactttgag   4620 

ttgagagcaa agacacgcag tgcaaactgg agctcaaaag ccaggttatc agtttattgt   4680 

gaagaagtga agacaaactt tgaaactaga tcagacagac tttgtgtcag tgtttgcaca   4740 

cgacatcatt tgcaagacat ttacacaaaa catctattaa aaaggtttgt tcatcctcta   4800 

gatgacttga ataacactac aaagtggaaa cataaagtga cacttcagtg tcttttcatg   4860 

atctgttata acttcagaat taatataaaa cttaatcatc tttcccaaat ggatgcgcct   4920 

taaaaatgtg tcacatttta agagaaataa actggttttc caaccatatg agatctatta   4980 

tcaaaacgta ttgtttcaac catgaaaaaa tccttttttt ttttggacta taagtcgcac   5040 

tttagggaga aatgtacctt acaaaacatt gcagaatgaa tggggatttt atcttgagag   5100 

acaaataata aaagaatagg taaaaatgac agactggata tgttcctccc agtgttttcc   5160 

cgcactgact gggttgtttg ggtggaaccc gctgccgctt ggacgggttt tattttctgt   5220 

tattgggagt gttcaacagg gttttcacgg gtctctttca tccgcttcac tggtccgacg   5280 

gatcagccgc tgacagcagc atctcctctc ccacctccct tgtggccgac cacgtgtctg   5340 

cgcaggccct cctgggactt ggacgcctgt tgggactcgt gactatctac ccctccctgc   5400 

ctacctgcag tagtgactga ttgtactcaa gttcacttgt tgtctttttg tttctttaat   5460 

tttgtggtca cactctgcgt gtgagtgggg tattggttct gttttgggtt tgagtaccag   5520 

ctctgtttaa aaggtgggct tgaagctcct atagggtttt atttttttta ttttaatcct   5580 

gtttttaaat ctatttatga taaatctttt aaatttggaa tcacgtctcg acctgattga   5640 

gtccccattt ttttattatt gtgcctgaac tgctcgtgtt gccacatatg ctttactact   5700 

gtagcatatt gaggcttttt cacgaaacat agaaggaatg tgtcggatct gataaattct   5760 

accaaggccg gcatgttttc ttctgcgttg ctgtcagtag caaatagtga tacaaacacc   5820 

tacagcgccc tctattggtt tttgctatca caaaaaaagt cccactgggg tataagttgc   5880 

atctctggcc aaactatgca aaaaactgtg acttatacac tgaaaaaact agttcttaac   5940 

agaatttttt tatttgtgct gatttcatgt ttacccttct aatgttaatt tgtctatgga   6000 

catgatggat cggtttttcc ttttatgagt aaaaataatc tctgtttcaa tgtatttact   6060 

cttttaaaca cagacctagc tgcactaagg caaagtggtt agcatttttg cctcaaaacg   6120 

agaagtcctt ggttcaaatc ccagccagga cctttctatg tgaagtctgt atgttcttct   6180 

tttgcatgca ggtcactccg gctttcttcc acagtccaaa aatatgttta atagattaat   6240 

tgatgtttct aaattgcccc aaggtctgca tttgggacag gctgcaaacc tgtccaaggt   6300 

gtaccctgac tttgctcaac agtctggtac aggctcagac agccctgtga ttccgaaacg   6360 

ggtacagtgg gttaaaaaaa tgtgtggatg gatggaaacc cagtcacaca gaaatattgt   6420 

aaaatgagaa tagttttcaa gctaacagat cttttgttaa ctatataagt ttacatattg   6480 

gtaaattgta gcattttcat ccatttctat aagtgctaca ttcaaatcca catgaattag   6540 

aggaggtaag acatagccat agtatagtat tttttgacag ctgaaacacc tgtggataat   6600 

caatgaaaat gaaatttaaa aatggtgtct tcctgactgt atttgttttg ttattgtcga   6660 

tacgactggt gcgatgacaa atcgcttggc tctttaaagc aagtgacaag agccgcattc   6720 

ttcctcttag gaaaagtttt ttttaactgt ccttcactcg ttattacttc tttttctctt   6780 

cgttattggt ctaaatgaga taagagccgt ttcgttctcg accgacacat cacagttttg   6840 

ttcagtatgc ctttcctgtt gctgaagcca aaaataaaaa tgactggatt tttttctaaa   6900 

aaaaaaaata aaagctccaa aggcaggtgt ctcaaatttg atttattttg cgaacaaaat   6960 

ttcttttagt agttactttc atcagtttta ttgtatgtgg agtcatgggt catatgtttt   7020 

aacctgaggc tagtgggaag ggtgaagggt gggttgggtt tttattgtaa tattgtgtgt   7080 

gtgttttttt ttttttcttt tctaaatgtt aaagcgcttt gagttacgct atgtatgaga   7140 

agcgcttttc tgaaataaag aaataaataa aatcaatctt tatttctata gcacttttca   7200 

tataaaacaa aacaaaacac aaagtgcttt acaccagaga aaaaaaaact agccccaacc   7260 

cacacagaac ccctaatccc attcaagcct cccacccctt aaatgatgga tataaacatt   7320 

tgggaaagag gctgagtatg gacaaaaatt gtttgtgtaa aagttaatat atggaacctt   7380 

cctctctgtg aatagctgca tagacagcca tgcagcatca caggtgggga cccagcaaca   7440 

ccacagcaag gtggcgatgc aggttcccat ctgagccgca gcggcaaaac agcagcggaa   7500 

ccaaagggag aggatccaac tgaggaagct ctggaattta aaatgaagat aaaaataaaa   7560 

aataaagaaa acataattag taaaagaaag aaataattaa ccacaaaatg tgaaataagg   7620 

aatattcaaa agaattgtta gtcccgtaga aattcatata atttaatata gtggataaat   7680 

taataaatac aaaataaata aaaactatta aaatggtaaa attagctaaa agcctgttta   7740 

aaaacattag tcttgagcct ttccttaaaa gcaactattc tctctgcagc cctcaggtcc   7800 

tctggcagac tgttctataa acaacaacca tagtacttaa aagatgcctc tatgtaggtt   7860 

ttgcttttta cagtcggaat gattaattga ctagtcagag gatttcgggt tcaaacacac   7920 

gggttcaaac ttaactaaga gatctgacag ataggaaggc acaaaattgt tagtatctta   7980 

aagcaggagt gtacatatcc taaaatcaaa aaaactaatc ttcttctgaa tttaaaaaaa   8040 

tgtacgtggt tagccttaat ttaattatct caggataccc tcccgaatcc ggtaggtggc   8100 

aagattgcac gtggaaactt acaaaatgca aagaaaatta gacaattgcg gcgaagaaga   8160 

atcacaaaca gtgggcggag cgcaacgccc ttaactccgc ctacgtgagc cacaactcag   8220 

ctcgagccgg tggtgtgagc ggcctaatca aacaaacatg acacaggtgt gctcctcgtg   8280 

ccctcagggc tctgttactg atgtaggtat ttgtaaacgg acagctagag ctcagctgaa   8340 

aagaagtgta attctattcg aacgtagtct ttaaaaaaaa atgaaggtgc cagaggcgga   8400 

attaatgagc gacattctga agaggctgac gggagagtct gctctgccgc tgtactgctg   8460 

catcgagaag ttcaagcgcg agaggaacgg cctctacttt gtcgccgagg atttcactga   8520 

aaccgtcaaa aaaagagaaa tggtcaacgc caaggaaaga ctgagagtga gttcagttta   8580 

gagaccaaaa atgatccata tttctaattt aaaaacatta ttaaacaaag cgaatatcag   8640 

acataatatt tgttgtagta tgacagtaat taatgtcatt attgctcagt gcagaaagtt   8700 

tgggaacagt agtttcatta tcttcagagc ccctccgggt ccaggaggga cttggccttt   8760 

ttctcagtcc tcctttctca tcctgcactc gtctttgaga agccgatcta accatgccgc   8820 

gcatttggag cttctgtcta ccatcaacta tgcattgagc aaagacacct ggagaggctt   8880 

cagttcaaac attgtgttct tggtgtcaat ttgactggaa agcactacag cttttaattg   8940 

ctattttatg tatttaaaac ctacaatttt aaactttata attcagcttt tttgagtgaa   9000 

tactcttcca ttaaaaacca gctgtggttc atctttaata attctgaaat tctcaaaact   9060 

tttatcaatt tgatctaagt tttgtgtcgc acaatatttc caggctctgc agttatcacc   9120 

gagttactgc tggatatgaa atttgggcct actttctcta cagataagga acttgaacac   9180 

aatgttctcc cggctgaagc gcatgctgcc tctaatgcaa ccagacaaaa agccaagtaa   9240 

agttgataca ctcaaagcag ccactgaata cattcgactt cttcttgctg ttttgcggga   9300 

cactgaaaat gtaagactgt cctccggggc ccctgtcaaa tcacaaaata aatgctgcaa   9360 

caacaacgct gatatacaat gttgactgct gtcattttac aacttgtaga acaacactgg   9420 

gacggatttt ctaaagaatg caatcactta tggtcagcag gatggcttcg ccaatgacct   9480 

ctggagaatg gacgatgtga gtatttaaat ctgtggctga aatggtagtt ttaaccaaac   9540 

atgtacctta gtagcatcac ctttacatca ggtttagcct ggaaactctg ctccatcgtt   9600 

tggaccacat gtgtcaaagt cgaggcccgc aggctacatc cggcccgcca gatgatttta   9660 

tattattatt aatggctcag caaagtgtag cgctgataac atatttacta cagatctcac   9720 

aatacagcgc ttcagctgcc cgacgaacta taatggtggc atcattttca ctacagatcc   9780 

cagaatcagt agcccgcggt ttacatgggc agaatatctt gatcggatca atggtcgggt   9840 

taaaatttca ccccgtgcgc aagggatcag aaaacctttt tcccgattag aacaaacgtt   9900 

cccatggctc acactttcgg tcggaatgaa tcatttgggc atgcgcagta gtgttaaaaa   9960 

ctcccggatg aggaaatggg tcccgttcta aacaaacagc tgccacagac atttatttta  10020 

tgctacagct cagtaatata cgagacgatc ttccaaatgt gcttttatta atgtcatgaa  10080 

tagtatgaag cgcctgttcg ctcatcgttt tatttaattc gtgtgatcag tgctggaaga  10140 

gggttaggac aggctataaa cacaggctaa caacattagc ctgatggaca caaaatccag  10200 

gaccataaaa cgctgcaatc tgcattctgg ctgtatgtaa atgtctggga ataacatcag  10260 

tctttattta gtcgggacac aactggaaac acaaaaacaa gtgattattt aaacagtttc  10320 

taaggactga gcaacattta tttctgcttc ctcaaagccg ctgtgtgtgc tggcggttcc  10380 

tcctgagtcc tggagctgct aacccagagc ggcgggacgg cttgcagtct gaattctccc  10440 

acacgtaaca aaacaaaaaa aaacgcacac gtaacaaagc atcctcctcc cctcagacac  10500 

aaagttatta atccagctcc tctgctcact cgggtgccag tggaccgagt gagcatcggg  10560 

ggggcacaaa gcgctccttc cccggatctc cctcatgggg ggtgacagag aggggagagc  10620 

cagcggggcg agagcggagc ggcgcttttc acggggcctc cgcagagcag ctggtcggtc  10680 

tcgtatgcgc tccaaagctt tctgtcagcg aaacaccatc tatgcgtgac gtaaaccaga  10740 

gcagtagtga cacgcgatcg gaatgaccat ttacatgctc cacgatcgga taaacgatca  10800 

ggataaccca cttatctcga tcggaaagaa attctgatcc gaacgagtct gatcggggca  10860 

gactattccg aacggcgcgt ttacatgacc cattttcttt ccgatcaggc gttctttccc  10920 

catgtaaacg cagctattgt tcagctgccc tgctgaacac ttttgctaga cccacgatgc  10980 

acaagagaag ttaattctga aaactgagcc tttaaaaact ctttggaggc agaattaatg  11040 

tttactgaca ttcagtaaac ctgcatgtca tctgtggagc gaatgtggct gtaatgaaag  11100 

aatatactct aagatggtgc tatgagacag atggcaaata cactttttac tgaaaagctg  11160 

agcacgtgga gtttgcacag cgctctagta tcattgaaga acaaaaaatg aattttgttt  11220 

tgtttgtaac ctgctttccg tcaatgtgga aacggcacct gtaaggattc agatggcgct  11280 

gattgagtgt tttggtacac cgaaggcaaa gtacgacctt caggttcaca cctccgttcc  11340 

tgcagaaatg ccccagctcc gtctacatgc agcccgatcc ttgtgcatgt ttgggcgcac  11400 

gtttctgtgt gagaagctcc tctcagtgat gaaaactaac aaaacagcag acaggagtca  11460 

tctccctgat gaacctctac aatacaggac ttcacatcaa acacaaacca acttgacaac  11520 

aaatgatgcc aggcgtccac ctgacaaaat gagacaagag caaagaaatt tgactttatt  11580 

ttgcagaaaa gcacaaattt tatttatata tccaggtttt atttgttttg ttatgcagca  11640 

aatacctatt ttgaattttt gtagttgtga caggatatat ttttatggag aacaaaatat  11700 

ttcggtatat ttaagttttt ttctatgaaa tcagagtaaa gttttttttt ttatctttag  11760 

tcgttttact ttatttcaaa ggtgtatcat tttgacagtt tatgttttta tggagagaaa  11820 

atataaagta tttaaggttt aagttagctc aacccatata acccagatac acttcttttc  11880 

ttaaacatag atatgtggac tacttggacc acagaaccca tttctgatgt ttttgttatg  11940 

ctggtgtcac catatgggtg acatcagcat gggttcttgt gggtaatagg atttctgttg  12000 

tttagttcag ttattttttt ctgagtttcc actgttagag gaattactga agtcaaatta  12060 

cttgcagtca tttctgtttc ctttggttaa caagctgcaa gtgtagttgt cactattact  12120 

aaagcagatc tccaaaagca gttggcaaat tctaacaagt ttaattttta caactttctt  12180 

atttggtttt cttttagaat ttgactccct taaatctaaa atggatcctc aaattagtat  12240 

tttgggggtt ttgttgactg aaaacttatt tggtttgctt acttaacttg cagttcctga  12300 

acatctgagc atttaactgt tattttttat ctgtctgaca gttcttgaac ctgtcagatg  12360 

atcacatttg gaggatgggt tcaccatgcc agcagaacct gcagcagagg atggagacat  12420 

gactagactg gtgttgcagc attgtgtgat gcctgcatac cagttcatca tccaagtagc  12480 

gcctgatcaa gcttcggtaa gtaaacaatt aaaggcgtgg gacttgatgt tttaaggtgc  12540 

aatttgtttg catgaaatga caacgtatca tttaatcctt caacactgga cctttgagct  12600 

ccattgtcta cagaattcat atattttggt ggattctgaa gtggaaaaaa acggctttgt  12660 

gtcggtgtgc aactttttac cataatagtg tgtagcacaa agtgcatatc ggcggtgcac  12720 

agctaacata caaagccact ttttttctgt ctgaatcagc tgattcacac taaatggttg  12780 

aaaggtatgg cagatggtgt gttatgtagg tcttgctggc gatatctcca gtgttggagg  12840 

gttaaattgg cctttgaatt cattgaggcc aaaataaaat ttattaaaat taagtcgggc  12900 

ctgaactgga ataactagtg aacaaagtaa aaccataatc ttaaaataaa atatgaattt  12960 

atatcatttt aatcaattgt gaatgtgttt aaatcttttc agtggcagcc acgaatcaaa  13020 

ttctatacag tgatccctcg ctataacaca gtttactttt cacggtatcg ctacttcacg  13080 

gatttgcatc gtgcattgag ttctgcattc tgattggcta aaaagtcact ccccttcttc  13140 

tacctgtgca tcaataacgt tgcagtttaa tatgtgcacg tacgtaaaac agctcgccaa  13200 

atttacatta tgtacgtgca aattttcttt ctggtggcat gtcggtgtat aaggatcttt  13260 

tggcaaagaa gaaaaaagag cgacatctgc ctatcactac gttcttctcc ctaacaaaca  13320 

cagctgcacc gcgggcttca aaagaagaaa acactgcaga gcggagtcag gatgcagcgg  13380 

ctcagtctga agggcagttg aaatacacct gagtcactat ttgtcccgac tggaccttgg  13440 

aatttttttc ataatcattt taaaattttc tccagtttaa tcctattact cgggtccgcg  13500 

gg                                                                 13502 

 
           
             23  
             4067  
             DNA  
             Oryzias latipes  
             
               misc_feature  
               Genomic DNA for gene 8  
             
           
            23 

ttgccaattt tttttattat gttcaaagtt ggaaagaaaa acaaaataca ttttaatagc     60 

actttcattt gtgacatgaa aagctagaaa tttacagatt gcgagttatt tcaagtacag    120 

gcatgtttta ttgccacctt gtggactttt atggtaatga cctgccaggt ggacctaaac    180 

tggttcgatt aatatacagt ccataaaaaa cagtcataat caacaataag agtgaacagt    240 

aaacaatcat tggtctcttc tattcgcccc tgtctcgcgg tacatgcgca cttacagggg    300 

cggggagggg cccctgattg ctgagctcct acaggtgtgc ttgagcagca ggtttaatcg    360 

cctgcagctg ctcggactct tttcagtgcg cagggcctcg tcgctccttc caaactctga    420 

ggatggactg caacctgcgg ctggccgttt cttgctggat catggtcttg tcttgggttt    480 

cccctctgac agagagtcgc cagacgaaca gccgaggttc tacagaaagg cacttccaac    540 

ctccagtcgg gacgcacggc ggtctgcagc ctcggctcta ctcggtgaag cagcagccgg    600 

ccccggaggt ccccgaacag caccgccccg tcacggtcat atgccacccg gattccatgg    660 

aggttgtggt gaaggccgac atgtttgaaa cgggcctgaa tgtggacggt ggacatctgc    720 

gactgggttc caacactctg ggcgcgggcg gtgagtgcgg ggcggtccag aaaggagagg    780 

acgaattcac catctgggcc ctgttgtccg actgcggaac caaactctca gtaagtttcg    840 

aacacagcga gcatgcgcct aatgagtcca gcatgaaaag actgtcttct tagtcaacag    900 

aagagaagat catttattcc aacgttctga tctactcacc cgaaccttct gctgatgggt    960 

tgttaagatt ggaagctgca actattccag ttgaatgtca ttatgacagg tgagtcctgc   1020 

gtgcatattt atacgcacac ctattttgtg caacagtggc cctatggaag agtgtatgcc   1080 

ctgagactag aacgtcgtga gcctcaaatc catgagtcat actggtaccc aaatcctacc   1140 

tgcttgactc tcagaatcaa gggattggat tgggggttta aactgccaaa tggttcccaa   1200 

taatggctgt ctctgcagct tcccatttcc catgcctggc tgtattgacg actaatggaa   1260 

cattaacatt aaattttatt ttctctatag gagatactct gttgatggca tttcccttaa   1320 

atcaacttgg gttccctctg tctccacaac ttctgtgaac gaccagatag atttcaatct   1380 

gaaactcatg actggtaatc aggggtggct ttggaaatta tattttgtct gttcaaagcc   1440 

aatatgcggc ttcaacaccc tgattgtttc aattgcacag gtgactggca gtctgagagg   1500 

gagtcttaca catatttcct ggctgatccc attaattttg aagtctctgc catagtggaa   1560 

aatcacgtcc ctctgcgggt gtatgtggac cactgtgttg ctacggcaac tcctgatgca   1620 

gaggctaatt taagatatga atttattgaa cataaggggt gagcttaaag tcaagcattc   1680 

tgaaagttac ttttttttgc ctttattact catctgacat ttctgccaaa ctagctgcct   1740 

cgttgatgct taccttacaa actccggagt acgtttccta ccaagaaccg aggaacataa   1800 

actgaggttt cagctggaag ccttcaggtt ctatcaagaa cccagcaacc aggtgtggct   1860 

ccaaatgaaa catttgtacg cttatgaaag ttttcatact gcttaaccat ttttgtcgtt   1920 

gcacaacaca aggtaactcg gacactttgt gaactcaaat cttggcgttc tgcgtagttc   1980 

cccctttggc atatcccttc agggggcgcc acagcaaatc agccttcact gagtcacaca   2040 

ggtggtttgg cagagtttta tgccagatgc ccttcagaca accatgcatt tttacctggc   2100 

ccatttttat ttgctggtta tagttcagca gtagcgcaaa gggtcttgcc cacggaccca   2160 

tgctggatga agcttattgt gttccatggg aattgaaccc tgattccccg tgtgccttaa   2220 

gcaattggac gatcagtcac tatagtcggt ttaattattg tgcttgatcc aaaattattg   2280 

ctgactggtt tgggaaaact gcttgtgagt caccactttt ccctattgtt tccagattta   2340 

cattacttgt gctgtgaagg ctgttcctgc tgtacaggcg gtcagttctc agaaccgagc   2400 

ctgctccttt attgagaaca ggtgatctat tgaaatagaa atgcaaaaat gttctagtgc   2460 

ttgttgcaca aatcttaact gctaccatct cgctccagat ggcaatccat agacggtggt   2520 

gatcaggtgt gcagaagctg tgacgtgtcc aggcggggtc aggaaccgca agctgtgcca   2580 

tctcctaaaa tggcagtgaa cgccaaagac caaatcggtc tttcacagaa aaatatagtc   2640 

cacaataaag ccgagcatca accggcttct tacgtccatt tttggccggg agcatatcag   2700 

agccatcact ccaaacctca gcagtccacc aacagattta tgaagaggga tgctgacaac   2760 

aaatttcgtg agtagaactt aaagggccta tttcatgcaa ataaactttt tgagctttta   2820 

aattgttagt tcctcacaaa aaacaactcc aaagcagtat tttgctacag tcacgcattt   2880 

ctgagcattc ctttaaaaac ctgctctgag caccagcccc tcccaatcca caaaaacaca   2940 

ttgtgagcga ggaacagccc cttccatgaa gagtctgcgc tgccagcacc gcccccaggc   3000 

taacccacac ctactttata catggagcta gcgttggttg atcggcaaaa acgtttttat   3060 

tgtcatatgc tcagttgtct ctgcaaaagt tcagacgttt tcgtgggcga aacagacatg   3120 

ctgcggctct aggttgattg tgaaaagggc ggcacccaca cggagtgaaa gtcgttgcat   3180 

cagatcgagt attacttctg ggtaggaaaa tgatctgaga aaatgactaa tttcataaat   3240 

ttgttttttt ttgagtctgc taaaggttat acatatggat acacttgact ctttaaaggc   3300 

ttgataaaag catgaaatgg ccccttcaat taaaataaat ctgaagagtt tgtgttaatt   3360 

taatgtttgt ctattgtaaa agctggaaag tatctaaaat tgactcttgg ttttgaaaat   3420 

gtcttacttt gcagatcaag ctgtccaact ggggcccctc gttgtgctac cgtcaagaaa   3480 

agtagtttca gtggcaacaa atttttcaac atggtcagaa aagaacacct cctgagacct   3540 

ggaacacctg atcgaggagt ctgagttcat ttgagaaatt tggtcttgat gggctaaaga   3600 

attttgctgc aatccaataa attaataaat ttctaatgta aactttattt gtaaaaatga   3660 

tgctgcttat tctgtggacc actctttctc cagtaaatat tactggtttg tgtccccttt   3720 

aaatgtatat aatatcgact aaagcaattt gtgtggagaa gctgttggtc catggacttc   3780 

ataaaactaa agcgaacaac ttcattgtta ttgtatatca ggcacttggc ctcattaaac   3840 

ttttggaaca taactagata ttggattatg acaaattgaa gacgactgga gaaacgtgat   3900 

cctagctcat gtctcttgca gggacatgct attagggagc atttccttcc attactgcca   3960 

cacctaatca tgtagtcaca gtattgatta tctgaaggct atgatgtaac gtgtttgctt   4020 

agcttgagtg atcaattaca gctttgcctt aattattctg gaacctt                 4067 

 
           
             24  
             23  
             DNA  
             Artificial  
             
               PCR primer 863.3  
             
           
            24 

gtacaagcgc gagaggaacg gcc                                             23 

 
           
             25  
             24  
             DNA  
             Artificial  
             
               PCR primer 863.1  
             
           
            25 

ttctccagag gtcattggcg aagc                                            24 

 
           
             26  
             20  
             DNA  
             Artificial  
             
               PCR primer 1/15  
             
           
            26 

gatcaggcgc tacttggatg                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial  
             
               PCR primer 6a  
             
           
            27 

ggagatactc tgttgatggc                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial  
             
               PCR primer 6b  
             
           
            28 

cgtcacagct tctgcacacc                                                 20 

 
           
             29  
             18  
             DNA  
             Artificial  
             
               PCR primer 8.3  
             
           
            29 

agactcctcc atcaggtg                                                   18 

 
           
             30  
             21  
             DNA  
             Artificial  
             
               PCR primer F1  
             
           
            30 

tccttccctg tttcgtcttg g                                               21 

 
           
             31  
             21  
             DNA  
             Artificial  
             
               PCR primer R1  
             
           
            31 

ttgcaggtgg attcacagca g                                               21 

 
           
             32  
             21  
             DNA  
             Artificial  
             
               PCR primer F2  
             
           
            32 

gctttcctgc gactatcagc c                                               21 

 
           
             33  
             21  
             DNA  
             Artificial  
             
               PCR primer R2  
             
           
            33 

atttggatcc catgcaacca g                                               21