JP2005160359A - Method for determining whale species using insertion polymorphism of sine as index - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining a whale species by a SINE (short interspersed element) method. <P>SOLUTION: The method for determining the whale species comprises making each genome DNA library from one or more whale species to be determined and isolating a clone containing an orthologues gene locus into which a SINE family or subfamily is specifically inserted in at least one kind of species among the whale species, respectively. In the gene locus, the gene locus sequence in each of the whale species is amplified by PCR (polymerase chain reaction) by using a set of a PCR primer annealing flanking sequences positioned at both ends of the SINE family or subfamily. The obtained PCR product is subjected to gel electrophoresis and the whale species is determined by presence of a band exhibiting the existence of the gene locus into which the SINE family or subfamily is inserted. The gene locus sequence and the primer sequence useful in the method are obtained. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for discriminating whale species by the SINE method.

  The automation of DNA sequencing and molecular cloning techniques has dramatically expanded access to the genome of an organism. In addition, the flood of digital genomic information has attracted many biologists to bioinformatics using computers. The results of comprehensive efforts such as the Human Genome Project have shown in great detail what was revealed about 30 years ago by nucleic acid regeneration studies. That is, the majority of eukaryotic genomes are composed of repetitive elements that do not encode specific protein products and have no apparent function (Kazazian et al., 1998). As a result of genome analysis, the so-called “junk yard”, which does not encode protein, is newly introduced as a dynamic molecular “jungle” filled with active elements such as DNA-type transposable elements and RNA-type transposable elements. Recognized. Retroposons are an interesting ubiquitous repetitive sequence that is thought to be used as a taxonomic tool for phylogenetic reconstruction and population analysis, and is a member of the RNA-related transposable element (Brosius, 1991; Shedlock and Okada, 2000). Thus, retroposon diagnosis provides an important bridge between the relevant sub-fields of evolutionary biology at the individual or species level.

  The term “retroposition” refers to how the element moves between a parent locus and a target locus in the genome by an RNA intermediate (Rogers, 1983). This copy-and-paste process creates a backflow of genetic information from RNA to chromosomal DNA (Weiner, et al., 1986, Schmid, 1996). It differs from DNA-type transposons that jump around the chromosome, such as mariner and P elements, in a manner that does not leave an original copy at the parent locus, that is, a cut-and-paste manner (Clark and Kidwell, 1997; Hartl et al, 1997). An important feature used to classify retroposons is whether they encode reverse transcriptase (RTase), an enzyme essential for self-amplification (Okada, 1991; Eickbush, 1994). Among the retroposons that do not self-amplify, there are many short interspersed elements (SINEs) in the genome, which is considered extremely useful for eukaryotic taxonomy studies. . SINEs range in size from 70 to 500 bp and are classified into two categories, those derived from tRNA and those derived from 7SLRNA. The 7SLRNA-derived SINE contains only two SINE families: the primate Alu family and the rodent B1 family. All other SINEs examined so far have been shown to be derived from tRNA. LINEs encoding RTases for amplification also fall into two categories. A mammalian L1 that requires only a simple poly A sequence for reverse transcription without the need for a specific sequence for amplification, and a strict 3 'end to be recognized by RTase for amplification. Other categories, including most LINEs, that require unique sequence motifs. Most non-mammalian SINEs and LINEs share the same 3 ′ terminal tail sequence, and their presence allows SINE to be amplified by the LINE-encoded RTase (Ohshima et al, 1997; Okada et al, 1997; Terai et al., 1998). In the case of mammals, SINEs, including those derived from tRNA and 7SLRNA, appear to be amplified with the help of RTase encoded by mammalian L1. Therefore, in this case, the above conserved sequence motif is not observed in the 3 'end tail of SINE. A general diagram for the SINE retroposition is shown in FIG.

The fact that a copy of 10 ⁴ or more facts and SINE referred retro position always occurs in the unidirectional interspersed throughout the host genome, when it SINE insertion analysis is a powerful new approach to molecular taxonomy Standing up. This SINE insertion analysis complements other forms of character data, most notably DNA sequence and morphology analysis (eg, Murata et al, 1993, Shimamura et al., 1997, Takahashi et al, 1998). Nikaido et al, 1999). Papers on SINE evolution as taxonomy tools and their importance are available (Weiner et al, 1996, Deininger and Batzer, 1993, Schmid, 1996, Rokas and Holland, 2000; Shedlock and Okada, 2000, Shedlock et al 2000).

By the way, in distinguishing whale species, after amplification of specific regions of mitochondrial genes by PCR, they are electrophoresed on agarose gels to confirm amplification, and then the base sequence is determined using a sequencing method. Thus, species discrimination has been made using the difference in sequence as an index. Since the sequence method experimental equipment is expensive, the analysis is currently almost done by specialized companies. Therefore, in order to discriminate the species for a huge number of individuals, the number of analyzes is naturally huge, and the cost is great. Furthermore, base sequence analysis is still insufficient and uncertain as described below, and their interpretation is often regarded as a problem.
JP 2003-009866 A Shimamura M, Yasue H, Ohshima K, Abe H, Kato H, Kishiro T, Goto M, Munechika I, Okada, N. (1997) Molecular evidence that whales form a clade within even-toed ungulates. Nature 388: 666-670 . Nikaido M, Rooney AP, Okada N. (1999) Phylogenetic relationships among cetartiodactyls based on insertions of short and long interpersed elements: hippopotamuses are the closest extant relatives of whales. Proc. Natl. Acad. Sci. USA 96: 10261-10266. Shedlock AM, Okada N (2000) SINE insertions: powerful tools for molecular systematics. BioEssays 22: 148-160. Nikaido M, Matsuno F, Hamilton H, Brownell Jr. LR, Cao Y, Ding W, Zuoyan Z, Shedlock AM, Fordyce RE, Hasegawa M, Okada N. (2001) Retroposon analysis of major cetacean lineages: the monophyly of toothed whales Proc. Natl. Acad. Sci. USA 98: 7384-7389. and the paraphyly of river dolphins. Norihiro Okada (1997) Origin of Whales and Mammal Evolution-Did Mammals Disappear After the Dinosaur Extermination? Science vol.67 Iwanami Shoten pp.908-916. Norihiro Okada (2000) When whales were on land-Relationship with hippopotamus revealed by system determination by sign method Science vol.70 Iwanami Shoten pp.119-125.

  Therefore, there is still a need for a test method for discriminating cetaceans in a simple, rapid and inexpensive manner without using base sequence analysis.

The present inventor has recently found that SINE can be used not only for phylogenetic and taxonomic studies as described above, but also for a simple, quick and inexpensive assay method for whale species discrimination. As a result of intensive experiments on a wide range of samples, the certainty was proved and the present invention was completed. That is, as described in detail below, if SINE insertions are common, it is possible to clarify phylogenically that some of the common insertions are a single system, If the insertion of the SINE family or subfamily is unique within a particular species, it can be seen that the presence or absence of the insertion can be used for species discrimination. Methods for isolating and identifying such a species-specific SINE subfamily sequence are also within the scope of the present invention.
Previously, phylogenetic tree creation using the SINE family has been made, but it has been done without classifying it as a subfamily using the SINE family. SINEs that enable species identification can only be performed with recently amplified SINEs, so it is necessary to specifically isolate recently amplified subfamilies. The method provides that method.

In one aspect of the present invention, there is provided a method for discriminating whale species by the SINE method, comprising the following steps:
Create a genomic DNA library from one or more whale species to be distinguished;
Isolating a clone containing an orthologous locus into which a SINE belonging to a SINE family or subfamily is specifically inserted in at least one of the whale species from each of the libraries;
Using the PCR primer set that anneals to flanking sequences located on either side of the SINE family or subfamily at the locus, the sequence of the locus was amplified by PCR for each of the whale species; and obtained The whale species is discriminated by gel electrophoresis of the PCR product and the presence or absence of a band indicating the presence of the SINE family or subfamily insertion locus;
Wherein the orthologous locus is GRY20 corresponding to the sequence shown in SEQ ID NO: 31, BRY50 corresponding to the sequence shown in SEQ ID NO: 32, Superm28 corresponding to the sequence shown in SEQ ID NO: 33, and the sequence shown in SEQ ID NO: 34 Hump42 corresponding to the sequence, Nag18 corresponding to the sequence shown in SEQ ID NO: 35, SIR13 corresponding to the sequence shown in SEQ ID NO: 36, Sei35 corresponding to the sequence shown in SEQ ID NO: 37, BRY63 corresponding to the sequence shown in SEQ ID NO: 38, GRY8 corresponding to the sequence shown in No. 39, MNK31 corresponding to the sequence shown in SEQ ID No. 40, NM1 corresponding to the sequence shown in SEQ ID No. 41, SEM3 corresponding to the sequence shown in SEQ ID No. 42, and the sequence shown in SEQ ID No. 43 Corresponding sNR2, na102 corresponding to the sequence shown in SEQ ID NO: 44, SEQ ID NO: 45 The method is selected from the group consisting of Tuti 35 corresponding to the sequence, Tut i 35 corresponding to the sequence shown in SEQ ID NO: 46, Sp9 corresponding to the sequence shown in SEQ ID NO: 47, and Sp2 corresponding to the sequence shown in SEQ ID NO: 48. Provided.

In another aspect of the present invention, there is provided DNA having the sequence shown in any one of SEQ ID NOs: 31 to 48 or orthologous DNA that hybridizes with the above DNA under stringent conditions.
The PCR primer can be selected from the group consisting of SEQ ID NOs: 49-84.
In another aspect of the present invention, a primer DNA having the sequence shown in any one of SEQ ID NOs: 49 to 84 is provided.

  As described above, the effect of the present invention is that the work of determining the sequence can be omitted. First, if you have the sequencer-related equipment necessary for sequencing, you will need a capital investment of at least 15 million yen. In addition, for consumables, the cost for one-time sequencing is approximately 1200 yen, and when a large amount (several thousand samples) of specimens is discriminated, a huge cost is required. In the case of outsourcing, there is no need for capital investment, but the cost is 10,000 yen or more per sample. In addition, since it takes 5 hours or more after PCR and electrophoresis to perform sequencing, the SINE method can be greatly improved in terms of time.

  As for data analysis, statistical operations are necessary for species discrimination based on DNA base sequences, and the results often vary depending on the analysis method, which is ideal from the viewpoint of species discrimination. Is hard to say. On the other hand, in the SINE method, species discrimination is performed using the irreversible phenomenon of SINE insertion as an index, so that the result is clear and no special statistical processing is required, and the result can be interpreted at a glance.

  For the above reasons, species discrimination using SINE insertion as an index is superior to the species discrimination based on the comparison of conventional nucleotide sequences in terms of money and time, and its reliability is high. It can be said.

Definitions In the present specification, “SINE” is a short interspersed repetitive sequence having a length of about 100 to 400 bases in primary structure, and in order from the 5 ′ end thereof, a tRNA homologous region, A homologous region, which includes a region rich in AT. At both ends, about 5 to 20 base direct repeats (direct repeats), which are considered to be formed when SINE is inserted into the genome, are characterized.
In the present specification, the “SINE family” refers to members of SINE having similar sequences to each other, derived from the same tRNA.
On the other hand, in the present specification, the “SINE subfamily” is derived from the same origin (family) but has a characteristic mutation, so that it is amplified from the same ancestor member in the SINE family. A group member. Subfamily is used interchangeably with type.
In the present specification, the orthologous locus means that it is derived from speciation from a common ancestor having two loci. On the other hand, when two genes are generated by gene duplication rather than speciation, they are “paralogous”. The orthologous gene, not the paralogous gene, gives the information necessary for estimating the molecular phylogenetic tree.

In the present specification, “flanking sequence” refers to a sequence located on both sides of an insertion site when a SINE family or subfamily is specifically inserted into an orthologous locus. PCR primers can be arbitrarily designed within such flanking sequences.
In this specification, stringent conditions mean that template DNA is put into a hybrid bag, an appropriate amount of prehybrid solution (6 X SSC, 1% SDS) is added thereto, the hybrid bag is packed with a sealer, and incubated for 1 hour or longer. Discard the solution, add the hybrid solution (6 X SSC, 1% SDS, 1 X Denhart's solution, Carrier DNA (Shared Herring Sperm DNA solution)), and heat denature the probe for 3 minutes at 95 ° C. Add soup, seal the hybrid back, and then incubate overnight (about 15 hours) in a 42 ° C water bath. Lightly wash the membranes with an appropriate amount of wash solution (2X SSC, 1% SDS). The conditions under which the probe hybridizes with the template DNA when rinsed.

In this specification, “outside group comparison” means a species or species group that is assumed to be most closely related to the outgroup, that is, the target organism group or ingroup in the phylogenetic theory. A method for determining the polarity of a trait based on it. By including the outer group in the analysis, the position of the inner group root (the common ancestor of the entire inner group phylogenetic tree) can be determined. In the initial theory of bifurcation taxonomy, first, the polarity of the inner group's trait state, that is, the primitive trait state and the derived trait state are determined based on the trait state of the outer group, and then the pre-determined Based on trait polarity, group species that share a derived trait state into a single lineage group. Outer group comparison is widely used as the main method for determining the polarity of morphological traits. The rationale is called the principle of saving the least, and the hypothetical trait state (criteria for polarity discrimination in the inner group) at the branch connected to the inner group root is estimated from the outer group trait distribution in the most conservative manner. Because. Maddison, Donoghue & Maddison (1984) and Swofford & Maddison (1987) are the most conservative phylogenetic estimation of the inner group based on the outer group comparison, without the polarity judgment for the inner group and outer group. And proved to be logically equivalent.
Therefore, it has become possible to estimate the most conserved phylogeny based on branch taxonomy, especially from molecular data that is difficult to determine polarity, such as restriction enzyme cleavage sites and nucleic acid base sequences.

  In the present specification, “covalently derived trait” means sharing an apomorphy determined to be derived as a result of estimation of polarity using an outer group comparison or the like. The theory of bifurcation taxonomy argues that only shared derived traits provide information for estimating phylogenetic relationships. This is because it is possible to assume a direct common ancestor that gave rise to species that share the estimated derived traits. Therefore, the shared derivation trait is a clue to construct a single lineage group (more precisely, a complete lineage group). Here, if an inconsistency occurs in the trait distribution, the derivative trait state of some traits must be considered as homoplasmy evolved from separate branches rather than from a common ancestor. The validity of the hypothesis of shared derivation traits is verified by the consistency with the phylogenetic hypothesis selected based on the principle of least saving, that is, the bifurcation diagram.

  In this specification, “polarity” refers to the evolution direction of the transition order between the trait states of a trait. Thus, the polarity depends on the model of transition order. The order of transition of the trait state reflects the trait of the trait data used for lineage estimation. It is not uncommon for a qualitative morphological trait to assume a linear or branched transition order. On the other hand, much of the molecular data such as nucleobase sequences and restriction enzyme cleavage sites are unordered traits that cannot be ordered between trait states. Bifurcation taxonomy and evolutionary taxonomy require polarity estimation of trait evolution prior to phylogenetic analysis. As criteria for estimating polarity, outer group comparison or fossil records and ontogeny information are used, but the most widely used is outer group comparison.

It is important to understand how SINE has evolved in order to effectively use SINE for the evolutionary phylogeny estimation of SINE and the isolation / identification method and species discrimination method according to the present invention. SINEs can be classified into families based on sequence similarity and subfamilies based on characteristic nucleotide presence and / or deletion, as described in detail below. The fate of SINE once inserted into the genome is determined by the balance between various factors in the chromosomal environment (Schmit and Maraia 1992) and mutations that prevent amplification from accumulating in SINE. . Furthermore, since LINE-encoded RTase is necessary for the amplification of SINE, if LINE loses its amplification ability in the genome and dies, it also means the death of SINE in the organism. (Okada et al., 1997).

  In the past, two opposing models of SINE evolution have been proposed for how SINE amplifies within the genome: a master gene model and a multi-source gene model. In the master gene model (FIG. 2A), only one or several “master” loci are the source of all descendant copies, which have no ability to replicate on themselves. That's it. In this scenario, its amplification rate over time is completely dependent on the conditions and activity of the master gene. On the other hand, in the multi-source gene model (Fig. 2 (B)), since the offspring can also have the same amplification ability as the parent copy, it functions as a "multi-source gene" over the course of evolution. is there. In the latter model, the amplification factor is a function of the rate of increase or decrease in total copy number from all of the source genes. The master gene model was proposed by empirical evidence from rodent ID SINE (Kim et al, 1994) and early studies of human Alu repeats (Shen et al., 1991). However, with subsequent detailed Alu sequence studies and comparative studies from various other taxa, it is now most reasonable to interpret that most SINE family amplifications are caused by the multi-source gene model. (Matera et al, 1990; Schmid and Maraia, 1992; Leeflang et al, 1992; Kido et al., 1994; Takasaki et al. 1994; Shedlock and Okada, 2000). In practice, a subfamily characterized by the presence and / or deletion of characteristic nucleotides will represent the respective source gene.

  The period between the birth and death of SINE is the period of SINE activity in the host genome, and its lifetime is directly related to their use in taxonomy and as a tool in the present invention. If the average sequence dissimilarity between members of the SINE subfamily is small, it is reasonable to assume that the subfamily is quite young and is still actively amplified in the host. If the average sequence dissimilarity between members of the SINE subfamily is large, it is reasonable to assume that the subfamily is relatively old and already inactive or dead in the host. However, diagnosis of common ancestors between host taxa using SINE insertion is possible only within the active life span of a given subfamily. The basic principle of this SINE method will be further described in the “Procedure” section below.

SINE Insertion Kinetics and Feature Theory The key to why SINE is a powerful taxonomic tool is that they are irreversibly independently inserted into the host genome (Murata et al, 1993; Shedlock and Okada 2000). There is no known mechanism for specifically removing SINE from the genome, and the present invention has a very low probability that two elements are inserted into the same locus or precisely excised from within the same locus. One can consider the presence of a SINE at the same locus in two different taxa as a polarized derivative phenotype in its genome. This defines one branch group or single lineage group in the strict sense of Hennig's (1966) method that uses only shared derived traits, or shared derived characteristics, to construct a phylogenetic hypothesis. In this bifurcation group, known ancestral conditions, or the lack of SINE insertion at a given locus, uniquely identifies the outer group without having to establish characteristic polarity through a competitive method of creating well-known artifacts. (Hendy and Penny, 1989) (see Figure 3).

Methods for isolation and characterization of SINEs Below, strategies for isolating new SINEs from genomic libraries, their screening methods, clone sequencing and SINE family-to-subfamily characterization, representative host classifications The quantification of copy number in the group as well as the definitive diagnosis of the SINE insertion pattern that provides phylogenetic information is described.

procedure
Species selection method for generating a genomic library The SINE method consists of the following basic steps: 1) generation of a genomic library from the selected species; 2) isolation of clones containing the SINE locus; 3 4) the design of the DNA sequence of the clone; 4) the design of the polymerase chain reaction (PCR) primer within the flanking sequence of the SINE locus; and 5) the presence or absence of SINE between the relevant species of interest. Including diagnosis. Although the SINE method can provide deterministic results quickly and reliably once established, it requires considerable time and effort to establish and establish its procedures and conditions.

  For example, consider the case of determining the phylogenetic relationship between closely related species A, B, C, and D. An actual phylogenetic tree of the above kind is shown in FIG. In this case, if species D is then chosen as the host from which to isolate SINE, a SINE locus that provides phylogenetic information cannot be obtained. This is because Species D contains a SINE locus inserted in the common ancestor of the four taxa of interest and in older origins. The locus inserted into all four ancestors is shown as SINE3 in FIG. 4 (B). The pattern of the electrophoresis gel of PCR showing the presence (+) or absence (−) of SINE3 insertion is shown in the lower part of FIG. 4 (C). To isolate a SINE that provides phylogenetic information, one must select species A or B that can provide the three SINE loci, SINE1, SINE2, and SINE3 shown in FIG. 4B. I must. Each of these loci defines a branching group, or single lineage group that shares a common ancestor in the evolutionary history of these species.

Isolation of a new SINE family from a particular species When no SINE family is known in a particular species, eg, species A in FIG. 4 (A), the SINE family is newly isolated and analyzed from its genome It is necessary. There are two ways in which a new SINE family can be isolated from selected species. One includes whole genomic DNA transcription in vitro (Endoh and Okada, 1986), and the other includes genomic DNA sequencing of about 60 Kbp or more facilitated by a new high-throughput automated DNA sequencing method.

Whole genome DNA transcription in vitro Most SINEs are known to be derived from tRNA, and therefore SINE has an internal promoter for RNA polymerase III. Although SINE is transcribed very rarely in vivo, SINE can be easily transcribed from naked DNA in vitro. When transcribing certain total genomic DNA in HeLa cell extracts using radiolabeled precursor nucleotides, such as alpha P32-GTP, usually radiolabeled RNA is transcribed. In some cases, these radiolabeled transcripts form clear bands when they are subjected to gel electrophoresis (Endoh and Okada. 1986; Matsumoto et al. 1986).

  This radiolabeled RNA can be used as a probe to screen a genomic library from a selected species of interest. If this transcript forms a clear band in the gel, it represents the actual SINE family. This is because all of the same transcripts from each locus of a given SINE family aggregate to form a distinct band. Even when obscured bands arise from genomic DNA, they can be used as probes for screening. However, in the latter case, the transcript may represent multiple SINE families.

  From our previous experience, when 10,000 or more copies of SINE are present in the vertebrate and / or invertebrate genome, they are detected by in vitro transcripts of total genomic DNA. Can do. FIG. 4 shows several example patterns of transcripts from total genomic DNA from selected animal species (Endoh and Okada, 1986).

Sequencing of genomic DNA larger than 60 kbp by an automatic sequencer According to the inventor's experience, the number of copies of the SINE family in the genome is usually over 10,000. Assuming that the entire genome is 3 × 10 ⁹ bp long and the size of a SINE is 300 bp, such a SINE family will occupy 0.1% of the genome (eg, 300 × 10 ⁴ = 3 × 10 ⁶ ). Thus, by sequencing over 60 kbp, it would be possible to find two independent SINE sequences within this type of randomly isolated DNA fragment (eg 600 × 100 / 0.1 = ⁶ × 10 ⁶ ).

  This has been easily achieved in the laboratory with access to the latest models of high throughput automatic DNA sequencers. For example, a novel SINE family has recently been characterized from the elephant genome and this novel SINE has been shown to be distributed among all species of Afrotheria. This method can be applied to the genomes of all mammals and possibly most vertebrates.

Methods for accurately identifying the SINE family and deducing its tRNA structure After determining multiple copies of repeat units according to the method described above, they can be aligned and the consensus sequence of the repeat family can be deduced. In addition to the SINE element, there are many repetitive sequences in the genome, and it is essential to properly diagnose the sequence. Since most SINEs are known to be derived from tRNA, they contain a promoter for RNA polymerase III. The RNA polymerase III promoter is a conserved sequence block and has the properties of a first promoter and a second promoter separated from each other in its genome. This second promoter is highly conserved and can be easily recognized empirically.

Hereinafter, an example of CHR-2 SINE will be considered. The tRNA-like structure of these elements can be established as follows (see FIG. 6):
1. First, a consensus sequence of CHR-2 SINE is constructed from alignment of several sequences of CHR-2 (see FIG. 8).
2. The consensus sequence of the second promoter for RNA polymerase III is searched visually. This sequence is 5′-GT (or A) TCG (or A) -3 ′. There is no exception to this motif when screening this promoter. When this motif is present, it creates a stem loop structure containing this second promoter sequence. The number of bases in the loop is 7, and the number of base pairs in the stem is 5. Even when all of the bases in the stem region do not form a base pair, as shown in FIG. 6 (A), these bases are arranged at appropriate positions in the tRNA.

3. Consider a unit of 5 bases 5 ′ upstream from the stem region. This is because in protoplasm class ItRNA, this extra loop region consists of 5 bases. In the case of CHR-2 SINE, the sequence of this unit of 5 bases is 3′-CAGGG-5 ′. The 3'-PyPyPuPuPu-5 'sequence is typical for this extra loop in several tRNAs (Sprinzl et al. 1987) and this can deduce the tRNA origin of the SINE (Figure 6). (See (B)).
4). The next 5 bases that form the aminoacyl-stem region of the tRNA structure are considered other units. In this case, it is 3′-GACGT-5 ′ (see FIG. 6C).
5). The next 7 bases that form the anticodon-loop region of the tRNA structure are further considered as other units. In this case, 3′-AACCGTC-5 ′. The 3'-TC-5 'residue at the 5' end of the AA residue at its 3 'end is a good indicator of this SINE tRNA origin. This is because these bases are highly conserved in most tRNAs (Galli et al., 1981). This further supports the tRNA origin of this SINE (see Figure 6 (D)).
6). Usually, the next 5 bases are considered other units, and they should base pair with the 5 bases assigned to the anticodon-stem region (FIG. 6C). In this case, the sequence is 3'-CGTCT-5 'and only the first 4 bases match well with the anticodon-stem region partner. In order to align this unit correctly, a deletion is placed at the position of the first base 3 ′ of the anticodon-stem (see FIG. 6E).
7). Next, a stem and loop structure for the D region of this tRNA-like structure is constructed. The number of stem and loop bases is usually 4 and 8, respectively, but can vary by 1 to 2 bases, especially in the tRNA-like structure of SINE. Obviously, some base pairs following CHR-2 do not form meaningful secondary structures. In this case, focus on the first promoter area. The most prominent feature in the first promoter region is the presence of two Gs in that region. Other features are G at position 15 (according to the tRNA numbering system; the same applies hereinafter) and A at position 14 in this loop. A at position 14 is the first base in the loop on the 5 'side. Therefore, these bases are placed at the corresponding positions of the loop of this tRNA-like structure (see FIG. 6 (F)). It is well known that T, the first base in FIG. 6 (F), is highly conserved in all tRNA molecules.
8). The tRNA-like structure of CHR-2 SINE can be deduced by merging the sequence shown in FIG. 6 (F) with other sequences for this family (FIG. 7 (A)).
9. Next, the BLASTN program in the GenBank DNA database (Altschul et al. 1990) is used to search for similarities between CHR-2 SINE and actual tRNA. In this example, tRNA Glu is most similar to the sequence of CHR-2. FIG. 7 (B) shows the secondary structure of human tRNA Glu.

Methods for characterizing the SINE family into subfamilies Assume that a SINE family is characterized in the genome of Species A in phylogeny, and that it is not known when this SINE family was first generated during evolution. Further assume that this SINE family was first generated in the old common ancestor of all taxon of branch group Y in FIG. In this case, the copy of SINE present in the genome of species A includes the old SINE amplified at time t in FIG. 9 and the young SINE amplified at time u. When screening a species A genomic library with this SINE family consensus sequence, both the old and new SINEs described above can be isolated. Since only the phylogenetic relationships of species A, B, C, and D are sought, it is inefficient to examine all times of an isolated SINE amplification event. Rather, it is much more efficient to attempt to isolate the SINE locus that is amplified near the branch of species D and spans all branches of the four taxa, including branch group X in FIG.

  As mentioned above, SINE and LINE are believed to be amplified according to a multi-source gene model (Schmit and Maraia 1992; Smit et al. 1995). If a source gene is mutated and successfully amplified during evolution, this mutated source gene can be recognized as a subfamily within its corresponding SINE family (Britten et al. 1988). Jurka et al. 1995). Subfamilies are amplified at some stage of evolution. Therefore, if a subfamily is amplified only in the common ancestors of species A, B, C, and D, a copy of this subfamily can be used effectively to determine the phylogenetic relationship of these four branch groups. Can be done.

  The SINE family consensus sequence in Species A was established as part of the above procedure and allows the design of one PCR primer at the 5 'end of the SINE sequence and other primers in the conserved region near the 3' end. To do. The sequence amplified by this primer set includes the entire SINE sequence. This primer set can be used to amplify many copies of SINE by PCR using genomic DNA from species A. The PCR product of this reaction can be cloned and sequenced in appropriate vector DNA. At this point, sequencing 100 copies of SINE from the PCR product is not a difficult task. By aligning these sequences, characteristic nucleotides or possible deletions representing this subfamily of the SINE family can be identified (see FIG. 10). Characteristic nucleotides are defined as those that are coordinately altered at three or more nucleotide positions within a particular subfamily and that can be distinguished from random mutations that accumulate in a random manner within the SINE sequence during evolution. Is done. Only after successfully characterizing a subfamily based on the presence of characteristic nucleotides and often specific deletions can the taxonomic distribution of a given subfamily be determined by dot-blot hybridization or PCR It becomes like this.

CHR-2 SINE subfamily Figure 8 shows the CHR-2 SINE family originally characterized as present in the genomes of Cetaceans, Hippopotamuses, and Ruminants ( The alignment of a copy of Shimamura et al. 1997) is shown. It is easy to see that there are six subfamilies in CHR-2 SINE. Due to the presence of the deletion, FL (full length), MDI (central deletion I), MDII (central deletion II), and the shortest group were characterized. This shortest group can then be categorized into subfamilies of DT (deletion type), CD (whale deletion type), and CDO (whale deletion tooth whale specific). FIG. 11 shows the alignment results of these subfamily consensus sequences. SEQ ID NO: 1 is the upper consensus sequence, SEQ ID NO: 2 is the FL consensus sequence, SEQ ID NO: 3 is the MDI consensus sequence, SEQ ID NO: 4 is the MDII consensus sequence, SEQ ID NO is the 5DT consensus sequence, SEQ ID NO: 6 is the CD consensus sequence, and SEQ ID NO: 7 represents the CDO consensus sequence.

  FIG. 12 shows the results of dot-hybridization experiments using probes specific for CD, CDO, and other subfamilies, respectively. This result clearly shows that the CD subfamily is specific to the genome of the Whales (dental and sperm whales) and that the CDO subfamily is specific to the genome of the dental whale. Therefore, SINEs belonging to the CD subfamily are useful for estimating the phylogenetic relationship of the cetaceans, in particular the lepidopterous whales, whereas SINEs belonging to the CDO subfamily are for estimating the phylogenetic relationship of the tooth whales. Useful. The distribution of copies of the CDO subfamily also suggests a single lineage of dentition whales, including sperm whales, which was one of the most controversial points in mammalian taxonomic biology.

Franking SINE PCR
After isolating the SINE locus from species A belonging to the subfamily generated in the common ancestor of branch group X shown in FIG. 9 and determining their sequence, the presence or absence of an insertion at a given locus or PCR experiments can be performed to diagnose absence. A primer sequence is selected by looking at the flanking (adjacent) sequence. Care must be taken in the design of the primer for the formation of secondary structure folds and for tandem annealing between the upstream and downstream primers. This can be easily checked using a variety of standard software programs written to facilitate PCR primer design, commercially or available through the internet. The melting temperature of the oligonucleotide primer is set around 55 ° C. Therefore, the annealing temperature for PCR must be based on this temperature when optimizing the amplification of orthologous loci from species B, C, and D. Sometimes the accumulation of mutations in the primer binding regions for species B, C, and D inhibits efficient primer-template annealing during this reaction. In this case, the annealing temperature must be reduced to about 45-50 ° C. If the PCR product is not amplified by PCR with the first primer, new PCR primers should be designed with additional care regarding potential artifacts that may reduce the efficiency of the PCR. FIG. 13 schematically shows the principle of flanking SINE PCR.

  FIG. 14 shows an example of the PCR result. In addition, the results of hybridization experiments conducted using the same filter using two different probes are shown simultaneously. FIG. 14 (A) is a PCR pattern that provides evidence that sea dolphins are single lines. This is because PCR products from sea dolphins have the expected fragment size including the inserted element, while fragments from other tooth whales have the expected fragment size lacking insertion in Mago 19. FIG. 14 (B) shows a hybridization experiment using a SINE probe, while FIG. 14 (C) shows a hybridization experiment using flanking DNA at the above locus. This latter experiment was carried out to prove that the orthologous locus was faithfully amplified by PCR in species other than the dragonfly from which the locus was originally isolated and characterized.

Interpretation of PCR Data When examining relatively recently branched species, flanking sequences at the orthologous SINE locus are faithfully conserved and typically do not cause problems that hinder PCR diagnosis. However, when examining relatively old and branched taxa, PCR fails more frequently and makes it difficult to interpret experimental results. In unsuccessful PCR, SINE-minus data appears. That is, there is successful PCR amplification at a given locus indicating the absence of a SINE insertion. An unsuccessful PCR represents lost data and is encoded as is (eg, “?”) When performing a most efficient analysis of the SINE character matrix that encodes the presence or absence of insertions.
When there is an inconsistent insertion pattern between the independent loci examined, there should be ancestral polymorphism and subsequent incomplete lineage classification.

Distribution of SINEs in the mammalian genome In general, most mammals have large amounts of SINE. They are clearly specific to the eyes, suborder, superfamily, family, genus, or species based on the hybridization pattern between the species examined (eg, the distribution of CHR-2 SINE in FIG. 12) . Such empirical evidence indicates that the SINE family was newly generated in many ancestral mammalian strains. However, the generation mechanism is not fully understood. The reason for this large number of SINEs or retroposons in the mammalian genome may be due to the fact that RTase encoded by mammalian L1 altered its specificity of template recognition in common mammalian ancestors. . This allows recognition of the poly A tail required for retropositions present in many LINEs that strictly recognize the 3 'tail responsible for the formation of the stem-loop structure (Ohshima et al. 1996; Okada et al. 1997; Kajikawa et al.). Such a scenario could have allowed poly A-containing RNA to become a pseudogene via L1 RTase in the mammalian genome.

  FIG. 15 shows a recently proposed phylogenetic tree of mammals (Waddell et al. 1999; Cao et al. 2000; Nikaido et al. 2000). The mammalian SINE family that has been characterized to date is noted on FIG. Briefly, the oldest SINE family distributed throughout the entire mammalian genome is MIR (Smit and Riggs 1995; Jurka et al. 1995). The Alu family is clearly specific for the primate genome, but its distribution among closely related species, such as lemurs, has not been investigated in detail. Rodents B1, B2, and ID are specific to the rodent genome. The rabbit C family has been reported in the genome of the order of rabbits, but its distribution among related species has not been reported. The SINE family present in the genome of the cetacean, such as CHR-1, CHR-2, CHRS, CHRS-S, PRE-1, and Bov-tA has been examined in detail (Shimamura et al. 1997; Shimamura et al. 1999; Nikaido et al. 1999). CanSINEA was first reported from the Canidae genome (Minnick et al. 1992; Coltman and Wright 1994), but was later shown to have occurred during evolution within many other carnivorous genomes (van der Vlugt and Lenstra 1995). A horse SINE, named the ERE family, was reported and its distribution was examined (Sakagami et al. 1994; Gallagher et al. 1999). The bat SINE family was isolated by Borodulina and Kramerov (1999) and named VES, and other bat SINE families have recently been characterized (Kawai et al.). The elephant SINE family was recently isolated and shown to be distributed among Afrotheria species (Nikaido and Okada). Here, SINEs with different tRNAs or completely different sequences are distinguished as families, and SINEs that originate from the same origin but have a diagnostic mutation are distinguished as Type or subfamily. A certain SINE explosively increases its copy number at a certain time, and it is thought that the explosive amplification time and the time axis in the evolution of the whole organism are related to the relationship between their phylogenetic relationship and the distribution of SINE.

  Importantly, although many SINE families have been isolated to date within the mammalian genome, they have not yet been characterized to their subfamily structure. Therefore, according to the present invention, the subfamily structure will be revealed in the future, and a new mammalian SINE family or subfamily can also be acquired.

New amplification of SINE: If fixed and short-term species branching SINE is very newly amplified in the evolution of the genome and is not fixed between populations of one species, the status as a co-derived trait is unstable, And it should not be used for phylogenetic tree creation. However, such a SINE distribution can be used for population structure analysis (eg, Hamada et al. 1998). If species branching occurs in a short period, that is, before most SINEs are fixed between populations via genetic drift, ancestral polymorphisms and subsequent incomplete lineages are inconsistent Create an insertion pattern. This phenomenon, coupled with the irreversible nature of the SINE retroposition, provides a bias for using SINE to examine historical patterns of phylogenetic classification in explosively derived taxa. If ancestors have polymorphisms, and then SINEs are incompletely distributed to offspring, creating contradictory insertion patterns, use one of the phylogenetic tree-saving methods to save at each locus. It is useful to evaluate the SINE character matrix for the presence or absence of insertions. Although unfixed polymorphic SINEs are not useful for high-level phylogenetic relationships, they are known to be excellent taxonomic tools for population analysis (Deininger and Batzer, 1994, Stoneking et al, 1997; Hamada et al, 1998). Therefore, by identifying and characterizing the SINE family or subfamily according to the method described in the present specification, it is possible to perform an animal species discrimination method using such a SINE family or subfamily.

Function and value of flanking sequences (usefulness)
Information on nucleotide flanking sequences within the locus examined for SINE insertion is useful. Of course, the insertion data is useful for the creation of the phylogenetic tree and the method according to the present invention, but the integration of the insertion sequence with the flanking sequence is the length of the branch between the branch groups determined by the independent SINE insertion event. Provide information on the safety (Lum et al, 2000; Shedlock and Okada, 2000). Furthermore, since the flanking sequences associated with a given insertion element are literally linked, the consistency between the SINE-derived topology versus the flanking sequences is an evaluation of the basic assumption of irreversible insertion at each locus. Provides a number of approaches (Lum et al, 2000). If there is homoplasmy (genetically homologous) or trait contradiction at an independent SINE locus, there may be a clear discrepancy between the phylogenetic trees. Such an approach is beginning as a new dimension of SINE analysis and provides the basis for enhancing the statistical evaluation of the SINE method.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the technical scope of the present invention is not limited by the following examples.

Materials and methods
Buffers, enzymes and other reagents Various buffers used for the operation of nucleic acids, enzymes, medium used for culture of E. coli, and other reagents were purchased from Wako Pure Chemical Industries, Ltd., Sigma, Difco and FMC. The material was adjusted with reference to Sambrook et al. (1989). Various restriction enzymes, modifying enzymes, and vector DNA were purchased from Takara Shuzo Co., Ltd., Toyobo Co., Ltd., and Amersham Life Sciences. Radioisotope was purchased from Daiichi Chemicals. PCR primers used were those commissioned for synthesis by OligoExpress (Amersham Pharmacia Biotech Co., Ltd.).

Genomic DNA
Various samples (tissue or DNA) used in this study are shown below. The following table 1 shows the English name, Japanese name, scientific name, etc. of whales and cloven-hoofs used in the analysis.

Shown in
Bactrian camels extracted DNA from muscle tissue provided by Dr. Kei Yoshida of the University of Tokyo School of Medicine. For pigs, the DNA stored in our laboratory was used as it was. The peccary used DNA provided by Dr. Hiroshi Yasue of the MAFF Livestock Experiment Station. Java deer used DNA provided by San Diego Zoo, USA. The DNA provided by Mr. Isao Munechika of Chiba City Zoological Park was used for Axis deer, Amyme giraffe, Sable antelope, Japanese serow, Markor, Muflon and Hippopotamus. The sheep was extracted from the liver provided by Mr. Yasushi Mukai of Ueda Meat Sanitation Inspection Center in Nagano Prefecture. For cattle, DNA was extracted from the kidney provided by Sagami Branch, Kanagawa Meat Sanitation Laboratory.

  For narwhal and giant whale samples, we obtained tissue fragments from Dr. Masaki Miya of the Chiba Prefectural Central Museum and extracted and used DNA. As for the sample of the humpback whale, it is known that it is a genus of the blue whale family, but the species name is unknown. Other samples, except for dolphins, were extracted from various pieces of tissue provided by Dr. Hidehiro Kato of the Large Whale Laboratory at the Fisheries Agency. For dolphins samples, Dr. Robert L. Brownell, Jr .: Chief Marine Mammal Division, Southeast Fisheries Science Center, PO BOX 271, La Jolla, California 92038 We had you go through the procedure for obtaining. Based on this, DNA samples were obtained from Dr. Healy H. Hamilton of California State University Berkeley for the subcutaneous tissue pieces of Amazon dolphins and liver tissue of La Plata dolphins. The Ganges River Dolphin provided dried bone samples by Dr. Satoshi Yamada of the National Science Museum and extracted DNA. As for the Chinese dolphins, fresh blood was collected from individuals kept in the aquarium attached to the Institute of Aquatic Biology of the Chinese Academy of Sciences, and heparinized on the spot was transported to the laboratory and DNA extracted immediately. The extracted DNA is currently stored at a Chinese laboratory because it cannot be brought into Japan.

Genomic DNA extraction from tissue of animal species used for genomic DNA extraction analysis was performed by a simple method (Rapid method; a method using Proteinase K). The extracted genomic DNA was used as a library preparation and PCR template. They are also stored at 4 ° C. Cut the tissue (liver, muscle) of whales and cloven-hoofs stored in a -40 ° C fryer to a few millimeters with a scalpel, transfer to a microtube, weigh the tissue, and then add 1X to the microtube. 500 μl of TNE buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0)) was added. When the tissue piece in the tube is finely ground using a Pasteur pipette, the tissue and TNE buffer are diluted with 1X Lysis buffer (20 mg / ml Proteinase K, 2% SDS, 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10 mM). EDTA (pH 8.0)) was added, and the mixture was agitated by inversion, followed by incubation using a 55 ° C. water bath to completely dissolve the tissue piece. An equivalent amount of phenol was added to the solution, followed by stirring with a belly dancer (200-300 rpm) for 1-2 hours. After centrifugation (room temperature, 12000 rpm, 5 min), the supernatant was transferred to another 2.0 ml microtube. In the same manner, phenol / chloroform extraction followed by chloroform extraction was repeated, and 0.1-fold amount of 3M sodium acetate and 2-fold amount of ethanol were added to the final supernatant. At this time, in many cases, fibers were confirmed, and then rinsed with 70% ethanol, and then an appropriate amount of TE buffer was added to dissolve the pellet. Extraction of DNA from the blood sample of the Chinese dolphins was performed using a commercially available column (QIAamp Tissue / Blood Kit Cat. No. 29306: Qiagen Co., Ltd.).

Flanking PCR (mainly used for amplification of homologous loci)
Flanking PCR means that PCR is performed using genomic DNA as a template by designing primers in the vicinity of both sides (upstream and downstream) of the SINE sequence. The primer used at that time was designed to have a length of approximately 17 to 30 nucleotides and a Tm (Melting Temperature) value of approximately 55 ° C. Regarding the design, CPrimer (Ver.1.08, Bristol and Anderson (1995)), which can be downloaded on the Internet, was used as Macintosh free software.
Reaction composition: Template DNA (100-500 ng); 10 X PCR Buffer (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl ₂ 5 μl; dNTP Mixture (2.5 mM each) 4 μl; Forward and Reverse Primer (5 pmol / μl) 2 μl each; TaKaRa TaqTM (5 U / μl) 0.25 μl; adds ddH ₂ O up to final volume 50 μl.
Reaction conditions: 94 ° C (Pre-denature) 2-3 min .; and 30 cycles of 94 ° C (Denature) 30 sec .; 45 ° C-60 ° C (Annealing) 1 min; 72 ° C (Extension) 30-90 sec. At this time, the temperature of Annealing was changed appropriately depending on the Tm value of the primer as well as the ease of increase of the locus, and the optimum temperature was searched.

Colony PCR
Colony PCR is a very simple method for selecting whether or not a target DNA fragment is contained in a clone as an insert when subcloning is performed. In the past, plasmid DNA was prepared with Miniprep and then digested with restriction enzymes to confirm the presence or absence of the insert. However, it is possible to complete this process only by PCR, so it takes a lot of time and effort. It leads to shortening. First, the colony of E. coli growing on the plate is lightly picked with a toothpick and rubbed into a 0.5 ml PCR microtube, and then it is used as a template for normal PCR in a 20 μl reaction system. The primer used in this case is prepared based on a sequence specific to the vector. During the heat denaturation in the PCR reaction, the cell membrane of Escherichia coli is destroyed, and the vector DNA serving as a template is dissolved in the solution to enable this PCR. As will be described later, this PCR product can be directly used as a template for sequencing using the ABI sequencer after confirming the presence or absence of the insert, so it is possible to sequence the insert immediately after the insert check. Even a considerable shortening.

Determination of base sequence The following sequencer and sequence reaction kit were used to determine the base sequence. Sequence using LI-COR dNA Sequencer (Model 4000): SequiTherm EXCELL ™ II Long-ReadTM DNA Sequence Kit-LC (Cat. No. SE7701LC, manufactured by EPICENTRE TECHNOLOGIES); ABI PRIZM ™ 310 Genetic Analyzer Sequence used: BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (P / N 4303152, manufactured by Perkin Elmer).
When subcloned plasmid DNA was used as a template for sequencing reaction, one prepared by SDS-Alkaline and Mg precipitation method was used.
When determining the base sequence of the locus containing the SINE sequence isolated by screening, M4 and RV on the vector sequence, and the oligonucleotide primer (aloca (aloca ()) prepared based on the consensus sequence of SINE Manufactured by the same company).
For direct sequencing of PCR products, add Shrimp Alkaline Phosphatase (SAP) 2U / μl 0.5μl; Exonuclease I 10U / μl 0.5μl (both from Amersham Life Sciences) to the PCR product and incubate at 37 ° C for 30 minutes. After degrading the oligonucleotide, the enzyme was inactivated by incubating at 85 ° C. for 15 minutes, and used as a template for the sequencing reaction.

Genome library creation
Approximately 50 μg of sucrose gradient genomic DNA was completely digested with appropriate restriction enzymes (mainly Hind III was used in this study), extracted with phenol / chloroform and ethanol precipitated, and then dissolved in 200 μl of TE buffer. did. Create a 10-40% sucrose gradient in a 15 ml plastic tube for ultracentrifugation (Centrifuge Tubes-50 Ultra-Clear ™ Tube 14 X 89 mm, Order No. 344059: manufactured by BECKMAN) The solution was added, attached to the rotor, and centrifuged (25000 rpm, 15 ° C., 15 hours) with an ultracentrifuge (L8-70M, Serial No. 7C869: manufactured by BECKMAN). After centrifugation, 10-15 drops were added dropwise to a 1.5 ml microtube and fractionated (about 250-400 μl / fraction). The fraction was subjected to 0.7 to 1% agarose gel electrophoresis, 4 to 6 fraction tubes were selected so that the fraction containing about 2 to 4 kbp fragment was sandwiched, and ethanol precipitation was performed. Dissolved in. 0.7-1% agarose electrophoresis was performed again to determine the fraction used for library preparation.

Construction of plasmid library In this experiment, only a plasmid library using pUC18 / HindIII or pUC19 / HindIII was used, and no phage library was prepared. In our laboratory, we know that there are enough copies of SINE in the genomes of whales and cloven-hoofed animals, and that enough positive clones can be obtained without using a phage library. In order to save time, a plasmid library with relatively few operation steps was used. A plasmid library was prepared as follows. Composition: TaKaRa Ligation Kit Ver.1 solution A, 12 μl; solution B, 1.5 μl; Vector DNA (pUC18 or 19 / HindIII to 100 ng / μl), 0.5 μl; Insert DNA (Genomic DNA Sucrose Density Gradient Fraction), 1.0 μl: The above solution was mixed in a 0.5 ml tube and allowed to stand at 16 ° C. for 30 minutes or longer on a cool block. This genomic library is stored at -20 ° C.

The flow diagram 16 to clonal isolation including SINE sequences showing the flow of experiments in SINE method.
screening
Production of membranes for screening
A 0.5 ml tube was mixed with 1 μl of the aforementioned genomic library mixture and an appropriate amount of competent cells of E. coli (E.coli JM105 strain), and then allowed to stand on ice for 30 minutes. After heat shock at 42-45 ° C. for 45 seconds, the plate was plated on an L / amp / X-gal / IPTG plate that had been incubated at 37 ° C. and left overnight in a 37 ° C. incubator. The number of colonies per plate was adjusted to 200 to 300, and a library for 10 to 20 plates was spread. Colonies that grew on this plate were transferred to a nylon membrane (Colony / Plaque Screen (trademark) NEF-978: manufactured by NEN Research Products), denatured solution (0.4 M NaOH, 0.6 M NaCl), neutralized solution (1 M NaCl, 0.5 M Tris-HCl (pH 7.0)) in this order for about 3 minutes. The membrane was dried well after draining water. The plate after the colony transfer was incubated at 37 ° C. so that the colony was sufficiently grown again so that the positive clones described later could be easily picked up.

Preparation of probes used for screening Oligonucleotides or PCR products were used for screening. The oligonucleotide sequences actually used are listed in Table 2 below:

  Shown in First, in the case of oligonucleotide: ddH2O 27-37 μl; Oligo Nucleotide (5 pmol / μl) 1 μl; 10 X T4 Polynucleotide Kinase Buffer 5 μl; T4 polynucleotide Kinase 2 μl; [γ-32P] ATP 5-15 μl Was mixed in a 0.5 ml tube and incubated at 37 ° C. The sequences of oligonucleotides used as probes are shown in Table 2 above. When using PCR products (Primer-Extension method): Template DNA, 500 μg; Forward primer (12.5 nmol / μl) 2 μl; Reverse primer (12.5 nmol / μl) 2 μl dDTP Mixture (dATP, dGTP, dTTP) 2.5 μl The above mixture was heat denatured at 95 ° C. for 5 minutes, then annealed at 55 ° C. for 1 minute, and then transferred to ice. Further, the following reagents were mixed in order and incubated at 60 ° C. for 30 minutes. 10XBcaBEST ™ Buffer 2.5 μl; BcaBEST ™ DNA polymerase 2 μl; [α-32P] dCTP. Each probe after the reaction was purified by elution with NICK Column (Sephadex G-50 DNA Grade: Amersham Pharmacia Biotech Co., Ltd.). RI counts were measured with a liquid scintillation counter. (A Geiger counter was also used for simple measurement.)

The hybridization membrane was placed in a hybrid bag, and an appropriate amount of a prehybrid solution (6 X SSC, 1% SDS) was added thereto. The hybrid bag was packed with a sealer and incubated for 1 hour or more. Discard the solution, add the hybrid solution (6 X SSC, 1% SDS, 1 X Denhart's solution, Carrier DNA (Shared Herring Sperm DNA solution)), and heat denature the probe for 3 minutes at 95 ° C. The hybrid bag was sealed, and the hybrid bag was sealed, and then incubated overnight (about 15 hours) in a water bath at 42 ° C. Rinse the membranes gently with an appropriate amount of wash solution (2X SSC, 1% SDS). After measuring and confirming the count, washing was performed using a new wash solution with the water bath temperature set at 55-60 ° C.

After discarding the developer wash solution and rinsing lightly with a new wash solution, the count was confirmed using a Geiger counter. In the dark room, a cassette, a membrane, an X-ray film, and an intensifier were placed in this order, and this was placed in a −80 ° C. freezer to expose it. The X-ray film was taken out from the cassette in the dark room, and developed with the X-ray film in the order of developer, stop solution, and fixer.

To confirm the position of the positive clones in the manner to superimpose an isolated developing X-ray film and plates were seeded a library of Positive Clone. For a colony that seems to be a positive clone, before minipreping, a primer created based on the consensus sequence of the SINE sequence used for screening, not the vector sequence, to confirm the presence or absence of the SINE sequence in the clone Colony PCR was performed using Thereafter, a positive clone plasmid was prepared, and its neighboring region was sequenced using an LI-COR sequencer. Since the subsequent operations are as described above, they will be briefly described below.

  First, primers were prepared based on the flanking sequences, and flanking PCR was performed using genomic DNAs of various organisms as templates. And in many cases, it is possible to easily amplify homologous loci for closely related species of the species used for screening, but this is in contrast to species that are thought to have originated in distantly related groups or branches. For loci, more base substitutions in the neighboring region are accumulated, so annealing with a primer based on a single sequence used for screening does not work well, and it is often not possible to amplify by the first flanking PCR. It was. In that case, nucleotide sequences of various homologous loci amplified were determined, their consensus was taken into consideration, new flanking primers were prepared, and PCR was performed again.

  When estimating the phylogenetic relationship between whales and artiodactyla, Southern hybridization was performed after agarose gel electrophoresis to confirm whether the amplified band is a homologous locus. The process is shown below. After agarose gel electrophoresis, it was stained with ethidium bromide (EtBr) solution, and the electrophoresis pattern was confirmed and photographed with a digital camera. Then put one membrane fully soaked in denaturing solution (0.4 NaOH, 0.6M NaCl) and two filter papers in this order on the gel so that no air bubbles get in between, and then JK wiper, Kim towel (trademark), I put a dictionary as a weight and left it overnight (~ 15 hours). The membrane was neutralized by standing in a neutralization solution (1 M NaCl, 0.5 M Tris-HCl (pH 7.0)) for about 15 minutes, and then sufficiently dried. Subsequent operations such as hybridization and development are the same as those in the screening described above, and are therefore omitted. In addition, since the base sequences of most loci were determined at the time of estimating the phylogenetic relationships within the order of the cetaceae, basically confirmation by Southern hybridization was not performed.

Example 1: Distribution of CHR-2 subfamilies in the genome of the cetacean. Regarding the origin of whales, opinions are divided on the internal lineage by classification by morphology and molecular classification. The cetaceans are the three subspecies of the cetaceans and the cetaceans, including many species of modern life, and the original cetaceans (also known as the blue whales) that are thought to have become their ancestors. It is divided into eyes (Fordyce et al., 1994), and the classification of modern cetaceans is distinguished by whether or not it has teeth, as is clear from its classification name. However, teeth can be confirmed up to the very last stage of cetaceans, and some of the fossil species of the original cetaceans are naturally traits that appear to be in the middle stage of cetaceans and cetaceans. Since there is also a whale with teeth (that is, a whale with teeth), as a matter of fact, it is impossible to claim a single system of whales based on the existence of teeth. This is because, when considered systematically, the whale “髭” is probably a co-derived trait, but the “tooth” trait of a whale is considered a primitive trait. However, only whales have the ability to eco-location, and the presence of melon bodies associated with them is one of the most famous traits suggesting a single system of whales.

  While the morphological classification strongly insists on the single systematicity of cetaceans and cetaceans, phylogenetic analysis using molecules that have been carried out to date has made most of them whales alone. This suggests that no lineage group is formed. Among them, a major controversy was the phylogenetic tree proposed by the comparative analysis of mitochondrial gene sequences of Milinkovith and Meyer (1993). According to this analysis, the group of sperm whale superfamily included in dentinal whales It is said to be closely related to cetaceans rather than other cetaceans (ie, cetaceans are multi-system). This result was followed by a number of molecular statistical studies, many of which resulted in multiple strains of whales or insolvency (eg, Arnason et al., 1994; Adachi et al., 1996; Smith et al., 1996: see Figure 17). Therefore, we performed species discrimination using the SINE method to solve the following problems: Problem (1) Problem of phylogenetic monophylaxis; (2) Phylogenetic relationships among all dolphins, including all dolphins ; (3) Branching age of each whale. The present invention has made it possible to clarify not only the determination of system relationships but also the problems of statistical analysis that has been advanced with absolute reliability up to now.

  As described above, the inventor of the present application is roughly divided into CHR-2 present in the cloven-hoofed genome, FL (Full Length), MD (Middle Deletion type), DT (Deletion Type), CD (Cetacea Deletion type) The CDO (Cetacea Deletion type Odontoceti) subfamily was found to exist. Since each subfamily has a different amplification period in the evolutionary process, their distribution is considered to reflect phylogenetic relationships. The alignment of consensus sequences for each subfamily is shown in FIG. As is clear from FIG. 18, each subfamily has a diagnostic base substitution or deletion in them.

  In order to confirm the distribution of these SINE subfamilies in whales and cloven-hoofed eyes, dot hybridization was performed. For the probe used, an oligo probe was designed at a position where CD and CDO can be distinguished from each other (alignment bold line), and a PCR product was used for FL. The results of dot hybridization are shown in FIG. First, it was suggested that FL is distributed only in hippopotamus and cattle (representative ruminants) in all cetaceans and cloven-hoofed eyes, and is not present in the mammalian genome added as pigs, camels and other outgroups. Secondly, CD is distributed specifically only in the cetaceans and not in the cloven-hoofed eyes, and strongly supports the single system of the cetaceans. Furthermore, with regard to CDO, a strong signal was confirmed only in the dentition, so this subfamily was considered to have exploded explosively in the cetaceae subspecies, and the analysis of the sperm whale questioned from molecular analysis This data supports the single system. However, if the signal is not confirmed in the analysis by dot hybridization, the possibility that the number of copies in the line is simply small cannot be denied, and therefore the lineage relationship cannot be estimated only by the result. Therefore, we actually isolated the loci such that those SINEs were inserted into the cetacean genome, and estimated their phylogenetic relationships.

Example 2: Analysis of the internal line of the cetacean using the insertion of SINE into the genome as an indicator In the study on the internal line of the cetacean, it is distributed specifically among the cetaceans from among the SINEs existing in the cetacean Focusing on CD and CDO, which is thought to be specifically amplified in the order of dentition, we isolated loci containing SINE sequences belonging to these subfamilies from various genomic libraries of cetaceans. . The PCR primers used for the isolation of loci showing phylogenetic relationships are shown in Table 3 below.

Shown in

  The locus A (GRY20), the locus of gray gray (Bhale whale), the locus B (BRY50), the locus of Bryde's what (Bryde's whale), the locus C (Sperm28), the locus of the sperm whole (sperm whale) D (Hump42), humbback what (humpback whale), locus E (Nag18), fin whale, locus F (SIR13), blue hale, locus G (Sei35) The locus H (BRY) of sei whale (sardine whale), the locus I (GRY8) of Bryde's what (Bryde's whale), the locus J (Mnk31) of gray whale (the gray whale), minke The locus K (NM1) of the hale (minke whale), the locus L (SEM3) of the minke whale (minke whale), the locus M (sNR2) of the right what (semi whale), the right whale (semi whale) ), The locus N (na107), the narwhhal locus, the locus O (na102), the narwhhal locus P (Tuti35), and the baked whale locus Q ( SP9) was isolated from a genomic library of sperm whale and the locus R (SP2) of sperm hale (sperm whale), respectively.

  The nucleotide sequences of these loci were determined, the primers were designed in the region near SINE, the PCR reaction using the genomic DNA of the cetacean was used as a template, and the separation was performed by agarose gel electrophoresis. The results are shown in FIGS.

  The numbers corresponding to the lanes in FIGS. 19 to 21 correspond to the whale species shown in FIGS.

  As seen in FIG. 20D, for example, the locus named Hump42 confirms the insertion of SINE only in humpback whales. In other words, if a sample is analyzed using a primer that amplifies this gene locus, it can be easily determined whether the whale meat whose origin is unknown is a humpback whale. As can be seen in FIG. 19F, the SIR 13 can determine whether or not it is a blue whale. In addition, with Super 28 and BRY 50, it is possible to roughly distinguish whether the specimen is a cetacean or a bearded cetacean. Therefore, if experiments are performed using the primers shown in Table 3, it is possible to quickly and inexpensively perform highly reliable species discrimination for almost all types of whale meat on the market.

  In short, there are one set each of a PCR primer (A) for discriminating whether the specimen is a whale, a PCR primer (C) for discriminating whether it is a cetacean, and a PCR primer (B) for discriminating whether it is a bearded cetacean. . As a result, it is possible to roughly sort the specimens first. After that, if PCR is performed using a primer capable of discriminating a specific whale species, it is immediately known which species each sample corresponds to. Actually, 5 sets of PCR primers for discriminating cetaceans (N, O, P, Q, R) and 10 sets of PCR primers for discriminating bearded cetaceans (D, E, F, G, H, I) , J, K, L, M), in addition to the above-described three sets of primers (A, B, C), a total of 18 species discrimination primers may be used. In addition, since the number of whale species sold in the market is much limited, it is not necessary to use all of these primers, and it is possible to use these primers properly according to the purpose, making it possible to distinguish species more efficiently with a small amount of experiment. It is also possible to perform. In other words, if you only want to know whether or not you are a mink whale, one primer set is enough to distinguish. In terms of time, if the specimen is a tissue, the final conclusion can be reached in 3 hours and a half, 30 minutes for DNA extraction, 2.5 hours for PCR, and 30 minutes for electrophoresis on an agarose gel. As a result of conducting a pilot experiment, the correct answer rate was 100%.

Example 3: Creation of a phylogenetic tree inside the cetacean The SINE insertion patterns obtained from the results of Example 2 were summarized in a matrix (see FIG. 22). FIG. 23 shows a phylogenetic tree in the cetacea estimated based on this.

General diagram of SINE retro position. FIG. 2 (A) represents the master gene model and FIG. 2 (B) represents the multi-source gene model. It shows the usefulness of SINE as an indicator of evolution. Represents a phylogenetic tree model of species AD. FIG. 4C shows a PCR pattern. Figure 5 is an example of several patterns of transcripts from total genomic DNA from selected animal species. 2 shows a tRNA-like structure of CHR-2 SINE.

FIG. 7 (A) shows the tRNA-like secondary structure of CHR-2 SINE, and FIG. 7 (B) shows the secondary structure of human tRNA Glu. Fig. 4 represents the construction of a consensus sequence for CHR-2 SINE from several sequence alignments of CHR-2. A phylogenetic tree containing an old SINE amplified at t and a young SINE amplified at u. Represents the identification of characteristic nucleotides or possible deletions representing a subfamily of the SINE family by alignment. Figure 5 shows an alignment of the consensus sequences for the CHR-2 SINE family subfamily. The results of dot-hybridization experiments using probes specific for CD, CDO, and other subfamilies, respectively, are shown.

The principle of flanking SINE PCR is schematically shown. An example of a flanking PCR result is shown. FIGS. 14B and 14C simultaneously show the results of hybridization experiments conducted using the same filter using two different probes. 1 shows a phylogenetic tree of mammals. The flow of the experiment in the SINE method is shown. A phylogenetic tree proposed from molecular statistical studies (the controversy over the issue of phylogenetic phylogeny).

The alignment of consensus sequences for each CHR-2 subfamily is shown. The agarose gel electrophoretic diagram showing the insertion result of SINE of gene locus A-C and F-H. The agarose gel electrophoretic diagram which shows the SINE insertion result of gene locus D, E, K, and I, J, O.

The agarose gel electrophoretic diagram which shows the SINE insertion result of gene locus LN and PR. The matrix which put together the insertion pattern of SINE is shown. FIG. 23 shows a phylogenetic tree inside the cetacean estimated based on the matrix shown in FIG.

Claims (4)

A method for discriminating whale species by the SINE method, comprising the following steps:
Create a genomic DNA library from one or more whale species to be distinguished;
Isolating a clone containing an orthologous locus into which a SINE belonging to a SINE family or subfamily is specifically inserted in at least one of the whale species from each of the libraries;
Using the PCR primer set that anneals to flanking sequences located on either side of the SINE family or subfamily at the locus, the sequence of the locus was amplified by PCR for each of the whale species; and obtained The whale species is discriminated by gel electrophoresis of the PCR product and the presence or absence of a band indicating the presence of the SINE family or subfamily insertion locus;
Wherein the orthologous locus is GRY20 corresponding to the sequence shown in SEQ ID NO: 31, BRY50 corresponding to the sequence shown in SEQ ID NO: 32, Superm28 corresponding to the sequence shown in SEQ ID NO: 33, and the sequence shown in SEQ ID NO: 34 Hump42 corresponding to the sequence, Nag18 corresponding to the sequence shown in SEQ ID NO: 35, SIR13 corresponding to the sequence shown in SEQ ID NO: 36, Sei35 corresponding to the sequence shown in SEQ ID NO: 37, BRY63 corresponding to the sequence shown in SEQ ID NO: 38, GRY8 corresponding to the sequence shown in No. 39, MNK31 corresponding to the sequence shown in SEQ ID No. 40, NM1 corresponding to the sequence shown in SEQ ID No. 41, SEM3 corresponding to the sequence shown in SEQ ID No. 42, and the sequence shown in SEQ ID No. 43 Corresponding sNR2, na107 corresponding to the sequence shown in SEQ ID NO: 44, SEQ ID NO: 45 na102 corresponding to be arranged, Tuti35 corresponding to the sequence shown in SEQ ID NO: 46, selected from the group consisting of Sp2 corresponding to the corresponding Sp9, and sequences shown in SEQ ID NO: 48 in the sequence shown in SEQ ID NO: 47, said method.   A DNA having the sequence shown in any one of SEQ ID NOs: 31 to 48 or an orthologous DNA that hybridizes with the above DNA under stringent conditions.   The method of claim 1, wherein the PCR primer is selected from the group consisting of SEQ ID NOs: 49-84.   Primer DNA which has a sequence shown in any one of sequence number 49-84.

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