KR101480207B1 - Novel single nucleotide variation markers specific for white hair phenotype - Google Patents
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Abstract
The present invention relates to novel single nucleotide variation (SNVs) markers for white hair traits. More specifically, the present invention relates to a novel single nucleotide variation (SNVs) marker for white hair characterized by tyrosinase The amino acid sequences of tyrosinase, TYR and dopachrome tautomerase (DCT) were analyzed by interspecific sequence comparisons to determine the identity of Arg87Gln And single nucleotide variations (SNVs) markers specific for white hair traits including Thr509Ala mutations in the DCT amino acid sequence. SNVs markers according to the present invention are useful not only in the field of therapeutic development for novel target candidates of various albinism diseases including oculocutaneous albinism by providing genetic information related to the whiteness traits of animals, And can be used extensively for species improvement through control.
Description
The present invention relates to novel single nucleotide variation (SNVs) markers for white hair traits, and more specifically to amur (Siberian) tigers, bengal tigers (white tiger), snow leopards, African lions (TYR) and dopachrome tautomerase (DCT), which are melanin synthesis-related enzymes, in a variety of species including, for example, And to novel SNVs markers specific to the whiteness traits searched by performing interspecific sequence comparisons.
Studies of single nucleotide polymorphism (SNP) or single nucleotide variation (SNVs) through multiple-sequence alignment analysis have been conducted in recent years with the development of genome projects, It is widely used to identify genes.
Studies on the trait-gene association through comparative analysis of these sequences have been actively conducted in the field of developing SNPs or SNVs markers related to diseases or traits by comparing sequences of genes related to various individuals in the same species. For example, In Publication No. 2010/0303813, the SNPs of EGLN2 (Egl nad homolog 2) gene for anti-TNF responsiveness are presented; See Andersen J. D. et al., Forensic science international. Genetics (2011), e153-e154] show specific SNPs of the SLC45A2, SLC24A5 and TYR genes associated with Danish skin color; Korean Patent Laid-Open Publication No. 2011-0129783 discloses SNPs of CDK7 (CDK-activating kinase 7) and ESR1 (estrogen receptor 1) genes associated with breast cancer.
In recent years, studies on gene-trait-related markers specific to specific species have been conducted through a cross-species sequence comparison analysis, which shows evolutionary differences in the traits. For example, Kim et al., Nature (2011) , 479: 223-227 discloses a method for detecting a mouse rat specificity by using a de novo assembly sequence of a naked mole rat and comparing it with a mouse, a human, a mouse, a chimpanzee, SNCPs of UCP1 (uncoupling protein 1) genes related to thermoregulation are presented.
On the other hand, tyrosinase (TYR) is an essential enzyme for the synthesis of melanin. Mutation of the TYR gene of a cat is related to white color phenotype [Lyons, LA et al., Ani . Genet. (2005), 36: 119-126], mutation of the human TYR gene is known to be related to albinism including oculocutaneous albinism [King, RA et al., Hum . Genet. (2003), 113: 502-513; Opitz, S. et al., Hum. Mutat. (2004), 23: 630-631). In addition, the dopachrome tautomerase (DCT) and its DCT gene are known to be involved in the synthesis of brown eumelanin.
Therefore, the present inventors examined the genomic sequences of several different species including Amur tiger, Bengal tiger (white tiger), snow leopard, african lion, and white spider to search for SNVs markers related to the whiteness traits of animals, As a result of intensive investigation through interspecific sequence comparison with respect to the amino acid sequence of TYR and DCT related to the synthesis, Arg87Gln mutation (Arg27Gln mutation in SEQ ID NO: 1) in the TYR amino acid sequence inherent to the white hair- And that the Thr509Ala mutation in the DCT amino acid sequence is a SNVs marker specific for the white hair trait.
It is therefore an object of the present invention to provide novel SNVs markers associated with the whiteness traits of animals.
In order to achieve the above object, the present invention provides an amino acid sequence of an Arg87Gln mutation (Arg27Gln mutation in SEQ ID NO: 1) in a tyrosinase (TYR) amino acid sequence of a white bud and a TYR protein of a species other than a white bird The same mutation at a position corresponding to the mutation; And a group comprising the same mutation at the position corresponding to the mutation in the amino acid sequence of the Thr509Ala mutation in the amino acid sequence of the dopant chromosomal aberrant chromosome of the white rats and in the amino acid sequence of the DCT protein of a species other than a white rats Lt; RTI ID = 0.0 > (SNVs) < / RTI > specific to one or more selected white hair traits.
The Arg87Gln mutation (Arg27Gln mutation in SEQ ID NO: 1) and the dopachrome tautomerase (DCT) amino acid sequence in the tyrosinase (TYR) amino acid sequence inherent in the white fungus species of the present invention, New SNVs (single nucleotide variations) markers specific for white hair traits, including the Thr509Ala mutation in the genome, provide genetic information related to the whiteness traits of an animal, resulting in multiple albinism diseases including oculocutaneous albinism In addition to the development of therapeutic agents for new target candidates, as well as species modification through regulation of animal characteristics.
FIG. 1 is a view showing a total of five species including Amur tiger, White tiger, Snow leopard, African lion and White lion according to an embodiment of the present invention. And tyrosinase (TYR) amino acids of each species of human, mouse, rat, naked mole, and dog, whose genomes and sequences are known. And the sequences are compared and analyzed through multiple sequence alignment.
FIG. 2 is a view showing a total of five species including Amur tiger, White tiger, Snow leopard, African lion and White lion according to an embodiment of the present invention. Of the amino acid sequences of the dopachrome tautomerase (DCT) of each species.
Hereinafter, the present invention will be described in detail.
The term " (amino acid abbreviation) (number) "in the present specification means that the (numerator) th amino acid in the amino acid sequence of the protein is an amino acid (abbreviation of amino acid). For example," Gln87 " Means that the 87th amino acid in the amino acid sequence is glutamine.
The term " (amino acid abbreviation) (amino acid abbreviation) " (amino acid abbreviation) used in the present specification refers to the amino acid of the corresponding amino acid of the corresponding protein in the amino acid of the left side (amino acid) For example, "Arg87Gln" means a mutation in which the 87th amino acid of the amino acid sequence is substituted with arginine to glutamine.
The term "corresponding position" as used herein refers to a mutually mapped position when matching amino acid sequences of the corresponding protein between different species through conventional multiple sequence alignment .
As used herein, the term "same mutation" means a mutation in the amino acid sequence means that the amino acids before substitution are the same and the amino acids after substitution are the same. For example, the amino acid sequence of tyrosinase (TYR) Refers to a mutation in which the arginine at a position corresponding to the mutation in the amino acid sequence of a TYR protein of a species other than a white bud is substituted with glutamine.
According to one embodiment of the present invention, the genomic sequence information of all five species, including Amur (Siberian) tigers, Bengal tigers (White tiger), leprosy, African lion, Genome sequencing (NGS) method. Specifically, genomic library sequence information obtained from the tissues of Amur tigers was subjected to a conventional de novo assembly analysis method The gene sequence prediction using the scaffold sequence information was performed to obtain the genome sequence information of the Amur tiger, and then four other species except Amur (Siberian) tiger, The genomic library sequences obtained from tissue samples of tiger, snow leopard, African lion, Sorted (align) the sequence of the scaffold crotch species could be secured each genome sequence by a method for replacing the specific mutation (mutation) position.
In accordance with one embodiment of the present invention, a heterologous sequence comparison analysis was conducted to search for SNVs specific for white hair traits. Specifically, Amur (Siberian) tigers, Bengal tigers (white tigers) ( Tyrosinase ) or DCT (dopachrome mutant enzyme) genes, which are known to be related to melanin synthesis, based on the genomic sequence information of a total of five species including a lentivirus, The sequences are transcribed into amino acid sequences to obtain TYR or DCT amino acid sequences of each species and these are introduced into known human, mouse, rat, naked mole rat and dog ) With the TYR or DCT amino acid sequences of each species by means of a conventional multiple sequence alignment method.
As a result, in the amino acid sequence of the TYR of a white hairy white rat, the 87th amino acid was Gln (uncharged), while in the TYR sequence of other species not having a white hairy character, the amino acid at the corresponding position was Arg (positively charged) And the 509th amino acid was Ala (hydrophobic) in the amino acid sequence of the DCT of white, while the other amino acids in the DCT sequence of the other species having no white coat were found to be Thr (uncharged) Respectively.
On the other hand, Bengali-back was the case of the Tigers, even though the same as baeksaja have white fur trait and could not be found these mutations as well as other animals do not have a white fur trait, and therefore white fur trait of Bengal hundred tiger TYR (Tyrosinase) and DCT (dopachrome mutant enzyme) genes.
Therefore, SNVs (single (SEQ ID NO: 1) and single-stranded (SNVs) specific to the white hair characteristic including the Thr509Ala mutation in the DCT amino acid sequence, the Arg87Gln mutation in the unique TYR amino acid sequence of the white fur- nucleotide variations can be used as specific markers for single base mutations (SNVs) of TYR and DCT genes specific for white hair traits.
In the present invention, the amino acid sequence of the TYR protein or DCT protein of a species other than a white bird also has the same mutation at the position corresponding to the mutation, as well as the TYR and DCT gene specific to the white coat These mutations can be included in the scope of the invention since they can be utilized as specific markers for single base mutations (SNVs).
Accordingly, the present invention provides a method for detecting the mutation in the amino acid sequence of the TYR protein of the Arg87Gln mutation (Arg27Gln mutation in SEQ ID NO: 1) and the TYR protein in a tyrosinase (TYR) The same mutation at the position where it is located; And a group comprising the same mutation at the position corresponding to the mutation in the amino acid sequence of the Thr509Ala mutation in the amino acid sequence of the dopant chromosomal aberrant chromosome of the white rats and in the amino acid sequence of the DCT protein of a species other than a white rats Lt; RTI ID = 0.0 > (SNVs) < / RTI > specific to one or more selected white hair traits.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Example
: Specific to white hair traits
SNVs
Marker
Search
One) Animal tissue sample
Tissues from Amur (Siberian) tigers, Bengal tigers (white tiger), snow leopards, African lions and white horses, shown in Table 1 below, which were used for heterologous sequence comparisons, were obtained. Specifically, tissue samples of Amur tiger, Bengal tiger, African lion and African white hull were provided from the Everland zoo in Korea. The tissue samples of snow leopard were obtained from the original tissue sample (Mongolia export license No. 0390, Republic of Korea Import License No. ES2012-02507) from the Department of Veterinary Science, Seoul National University.
2) Sequence of genome (genome) for comparison of interspecies gene sequence
For intergeneric gene sequence comparisons, information on protein sequences of human, mouse, rat, naked mole rat, and dog, whose genome sequence is known, Were provided from UniProt's database [Kim, EB et al., Nature (2011), 479: 223-227; UniProt, Nucleic Acids Res. (2012), 40: D71-75]. On the other hand, in the case of all five species including Amur (Siberian) tiger, Bengal tiger, lepidopteran, African lion, and white porcelain whose genome sequence is unknown, genome sequence information was obtained as follows.
3) Amur (Siberian) securing the genome sequence of the tiger
In order to obtain the genome sequence information of the Amur tigers, a gene set prediction through a scaffold sequence was performed according to a conventional de novo assembly analysis method as follows.
First, to obtain genome sequence information of Amur tigers, three 170 bp (base pairs) and 500 (base pairs) sequences were generated using Illumina HiSeq 2000 (manufacturer: Illumina) according to a conventional next-generation sequencing short insertion paired-end libraries of bp and 800 bp long and long insertion mate-pair libraries of four 2 Kb (kilo bp), 5 Kb, 10 Kb and 20 Kb lengths, A total of 288.20 Gb (giga bp) dielectric libraries were obtained from 27 lanes consisting of mate-pair libraries, with an average read length of 76.62 bp. Based on this, a conventional method [Bairoch, A., et al., Nucleic Acids Res. (Scaffold) assembly software (SOAPdenovo) according to the manufacturer's instructions (2000), 28: 45-48) to obtain a total of 2.4 Gb of scaffold sequence information for the Amur tiger genome , Where the N50 value of the scaffold was 8.8 Mb (mega bp). Based on the obtained scaffold sequence, GLEAN [3] was constructed according to conventional ab initio prediction, homology-based prediction, and cDNA / EST-based prediction methods. Elsik, CG et al., Genome Biol. (2007), 8, R13], the genetic prediction was carried out as follows to obtain a gene set of Amur tigers.
i) ab initio prediction: HMM (hidden Markov model) -based repetition zone masking Generation of new predictions on repeat masked genome ( de novo prediction) was performed using AUGUSTUS (version 2.5.5) [Stanke, M. et al., Nucleic Acids Res. (2006), 34: W435-439] and GENSCAN (version 1.0) [Burge, C. et al., J. Mol. Biol. (1997), 268: 78-94].
ii) Homology-based prediction: homologous protein sequences of different species (from Ensembl 60 release) were obtained from TblastN (Blast 2.2.23) [Altschul, S. F. et al., J. Mol. Biol. (1990), 215: 403-410] (with an E-value cutoff of 1E-5). Aligned sequences and their specific proteins were screened followed by GeneWise (version 2.2.0) [Birney, E. et al., Genome Res. (2004), 14: 988-995]. ≪ / RTI >
iii) cDNA / EST-based prediction: Cat EST and full length cDNA (from UCSC) were amplified using BLAT (Kent, WJ, Genome Res. (2002), 12: 656-664] (blat-34, identity≥0.90, coverage≥0.90), and splice sequence alignment analysis was performed on the genome sequence. The EST results were analyzed using PASA [Haas, BJ , Nucleic Acids Res. (2003), 31: 5654-5666]. The splice sequence alignment was performed according to the overlap method.
4) securing genome sequence of bengal tiger, white tiger, snow leopard, african lion and white lion
Genus HiSeq2000 was screened in accordance with the next-generation sequencing method (NGS) for four other species except Amur (Siberian) tigers, namely Bengal tiger, snow lamb, African lion and white horses. Was used to perform genome sequencing to obtain an average 71.5 Gb long dielectric library sequence. These short readings were aligned on the scaffold sequence of Amur tigers using the program BWA (Li, H. et al., Bioinformatics (2009), 25: 1754-1760) Homozygous and heterozygous SNVs were searched using [Li, H. et al., Bioinformatics (2009), 25: 2078-2079] (with default option) Each genome sequence was obtained by substitution of mutation positions.
5) Intergeneric gene sequence comparison analysis
Based on the genomic sequence information of all five species including Amur (Siberian) tigers, Bengal tigers (White tigers), snow leopards, African lions and white horses, as described above, Gene sequences of TYR (tyrosinase) and DCT (dopachrome mutant enzyme) genes were searched and then transcribed into amino acid sequences based on amino acid codon tables, respectively. The obtained TYR or DCT amino acid sequences of each species were clustalW2 (SEQ ID NO: 2) with known TYR or DCT amino acid sequences of human, mouse, rat, naked mole rat and dog A comparative analysis was performed by matching through multiple sequence alignment using the program http://www.ebi.ac.uk/Tools/msa/clustalw2/).
As a result, as can be seen from Figs. 1 and 2, the mutation of the white hair-specific amino acid sequence of the white bud and its position could be confirmed. Specifically, as can be seen from FIG. 1, the 87th amino acid (27th amino acid in SEQ ID NO: 1) is Gln (uncharged) in the TYR amino acid sequence of a white hairy white rat, In the TYR amino acid sequence of other species including Amur tiger, African tiger, lily of the valley, human and dog, it was confirmed that all the amino acids at corresponding positions were Arg (positively charged). As shown in FIG. 2, In the DCT amino acid sequence of the wild-type DCT amino acid sequence, the 509th amino acid is Ala (hydrophobic), while the DCT amino acid sequence of other species including Amur tiger, white tiger and African tiger, All were found to be Thr (uncharged). On the other hand, Bengali-back was the case of the Tigers, even though the same as baeksaja have white fur trait and could not be found these mutations as well as other animals do not have a white fur trait, and therefore white fur trait of Bengal hundred tiger TYR (Tyrosinase) and DCT (dopachrome mutant enzyme) genes.
In conclusion, the Gln87 mutation (Arg27Gln mutation in SEQ ID NO: 1) and the Ala509 mutation in the amino acid sequence of dopachrome tautomerase (DCT) in the tyrosinase (TYR) amino acid sequence shown in Table 2 below Can be used as markers for single base mutations (SNVs) of TYR and DCT genes specific for white hair traits.
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
<110> Genome Research Foundation THERAGEN ETEX CO., LTD <120> Novel single nucleotide variation markers specific for white hair phenotype <130> NP12-1192 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 60 <212> PRT <213> White lion <400> 1 Ser Lys Ala Pro Leu Gly Pro Gln Tyr Pro Pro Thr Gly Met Asp Asp 1 5 10 15 Arg Glu Ala Trp Pro Ser Val Phe Tyr Asn Gln Thr Cys Gln Cys Phe 20 25 30 Gly Asn Phe Met Gly Phe Asn Cys Gly Asn Cys Lys Phe Gly Phe Trp 35 40 45 Gly Pro Asn Cys Thr Glu Lys Arg Leu Leu Val Arg 50 55 60
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| US20050266414A1 (en) | 2004-05-28 | 2005-12-01 | Regents Of The University Of California | Carrier tests for albinism in the cat |
| KR20100133249A (en) * | 2009-06-11 | 2010-12-21 | 대한민국(농촌진흥청장) | A molecular marker and genetic method for detection of albino in bos taurus coreanae |
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| US20050266414A1 (en) | 2004-05-28 | 2005-12-01 | Regents Of The University Of California | Carrier tests for albinism in the cat |
| KR20100133249A (en) * | 2009-06-11 | 2010-12-21 | 대한민국(농촌진흥청장) | A molecular marker and genetic method for detection of albino in bos taurus coreanae |
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| Title |
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| GenBank: AEO95503.1, tyrosinase, partial [Prionodon linsang], 2012.08.08. * |
| Peptides. Volume 26, Issue 10, 2005, Pages 1901-1908. * |
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