# Patent

## Title
Method for identifying a testicular cell of a chicken

## Abstract

The present invention relates to a nucleotide sequence of a testis-specific gene in avian species and a method for identifying a testicular cell of avian species.

## Background
CROSS-REFERENCES
The present application is a non-provisional patent application claiming the priority to U.S. provisional application Ser. No. 60/604,696 filed on Aug. 27, 2004. The entire contents of this application are incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a nucleotide sequence of a testis-specific gene in avian species and a method for identifying a testicular cell of avian species.
2. Description of the Related Art
Expressed sequence tags (ESTs) are short segments of deoxyribonucleic acid (DNA) that are generated from either one or both ends of expressed gene transcripts (Adams M. D., et al., (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651-6). Most of the ESTs were produced for genome-wide analysis using microarray technology and deposited in the dbEST (Boguski M. S., et al., (1993) dbEST—database for “expressed sequence tags”. Nature Genetics 4, 332-3) of the National Center for Biotechnology Information (NCBI). Currently, the dbEST is the most abundant source of new coding sequences. Up to Jul. 23, 2004, 22,830,690 ESTs from 721 species were deposited in the dbEST. In the species having relatively higher number of ESTs compared with its protein sequences, the EST data are probably more important resources for extracting biological and evolutionary information of the species.
Especially, the number of EST sequences in the chicken (Gallus gallus), which is currently reported 495,089, is more than 60-folds than that of protein sequences. Based on plenty of the chicken EST data, the institute for genomic research (TIGR) have produced Gallus gallus gene index (GGGI). The TIGR gene indices consisted of clusters of EST sequences and the clustered elements were evaluated to produce a set of unique and virtual transcripts with high fidelity, called Tentative Consensus (TC) sequences (Quackenbush J., et al., (2000) The TIGR gene indices: reconstruction and representation of expressed gene sequences. Nucleic Acids Research 28, 141-5). To date, TIGR has assembled the chicken ESTs with chicken transcripts into 43,866 TC sequences.
The testis is the organ that produces sperm, and during spermatogenesis transcriptional regulation within germ cells is carefully orchestrated (P. Sassone-Corsi, Unique chromatin remodeling and transcriptional regulation in spermatogenesis. Science 296 (2002) 2176-2178). Sperms develop in association with highly specialized somatic testicular cells, such as Sertoli cells and Leydig cells. During the differentiation of germ cells into spermatozoa, a complex paracrine dialogue with Sertoli cells occurs (M. K. Skinner, et al., Cell-cell interactions and the regulation of testis function, Ann. N.Y. Acad. Sci. 637 (1991) 354-363). Endocrine activity such as testosterone secretion by Leydig cells promotes germ cell differentiation (M. D. Griswold, The central role of Sertoli cells in spermatogenesis, Semin. Cell Dev. Biol. 9 (1998) 411-416). Thus, it has been speculated that the testis has specialized transcription complexes that coordinate the differentiation program of spermatogenesis. In birds, the female is the heterogametic (ZW) sex, but genes on the W chromosome do not influence gonadal development in the way that the SRY gene on the mammalian Y chromosome does. No sex-chromosome-specific SOX gene homologous to the mammalian sex-determining gene SRY has been found in birds (R. Griffiths, The isolation of conserved DNA sequences related to the human sex-determining region Y gene from the lesser black-backed gull (Larus fuscus), Proc. R. Soc. Lond. B. Biol. Sci. 244 (1991) 123-8). However, SRY-like HMG-box gene 9 (SOX9) may influence gonadal development by the initiation of transcription of anti-Müllerian hormone (AMH) during the early stages of chick gonad differentiation (E. Oréal, et al., Early expression of AMH in chicken embryonic gonads precedes testicular SOX9 expression, Dev. Dyn. (1998) 522-32). The avian DMRT1 gene is located on the Z chromosome (I. Nanda, et al., 300 million years of conserved synteny between chicken Z and human chromosome 9, Nat. Genet. 21 (1999) 258-259) and is expressed more strongly in male than in female embryonic gonads, (C. S. Raymond, et al., Expression of Dmrt1 in the Genital Ridge of Mouse and Chicken Embryos Suggests a Role in Vertebrate Sexual Development, Dev. Biol. 215 (1999) 208-220; C. A. Smith, et al., Conservation of a sex-determining gene, Nature 402 (1999) 601-2; Z. Shan, et al., Sex-specific expression of an evolutionarily conserved male regulatory gene, DMRT1, in birds. Cytogenet. Cell. Genet. 89 (2000) 252-7).
It is therefore thought that numerous genes affect male germ cell development in birds and, that some of these may be expressed in a testis-specific pattern. The chicken is one of the most important model organisms for the study of germ-line development, as its embryonic development occurs in ovo.
Throughout this application, various publications are referenced and citations are provided in parentheses. The disclosure of these publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.
SUMMARY OF THE INVENTION
Under such circumstances, the present inventors have made intensive researches to identify novel testis-specifically expressed nucleotide sequences involved in spermatogenesis in avian species, and as a result, discovered novel testis-specific nucleotide sequences in avian species.
Accordingly, it is an object of this invention to provide an isolated nucleotide sequence of a testis-specific gene in avian species.
It is another object of this invention to provide a method for identifying a testicular cell of avian species.
Other objects and advantages of the present invention will become apparent from the detailed description to follow and together with the appended claims and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents the proportion of ESTs collected from the Korean native chicken (KNC). Brain, spleen, and testis tissues were dissected from 26-week-old KNC and cDNA libraries were constructed from total RNA isolated from each tissue. 17,502 cDNA clones from the libraries of each tissue were sequenced and successfully read 13,132 sequences.
FIG. 2 represents the distribution of putatively new transcript (PNT) and PNT aligned against chicken genome draft sequences of the University of California Santa Cruz (UCSC) genome browser in each tissue. 1,176 putative novel transcripts were retrieved after the comparison and filtering, and mainly consisted of singletons (96%) in contrast with a small portion of contigs (4%). (A) The distribution of PNT singletons and PNT singletons aligned. (B) The distribution of PNT contigs and PNT contigs aligned.



DETAILED DESCRIPTION OF THIS INVENTION
In one aspect of this invention, there is provided an isolated nucleotide sequence of a testis-specific gene, wherein said nucleotide sequence is specifically expressed in a testis of avian species and comprises (i) a nucleotide sequence selected from the group consisting of SEQ ID Nos:1-25, (ii) its complement or (iii) its fragment.
The present inventors have identified testis-specifically expressed nucleotide sequences for elucidating mechanisms and processes underlying spermatogenesis in avian species. To demonstrate the expression specificity of nucleotide sequences in avian testis, the inventors have researched on various organs such as testis, brain and spleen and as a result, discovered novel testis-specific nucleotide sequences in avian species.
The testis-specific nucleotide sequences are differentially expressed in testis of avian species, preferably, a chicken, a quail, a turkey, a duck, a goose, a pheasant and a pigeon, more preferably, a chicken.
According to a preferred embodiment, the testis-specific nucleotide sequence consists of (i) a nucleotide sequence selected from the group consisting of SEQ ID Nos:1-25, (ii) its complement or (iii) its fragment.
The nucleotide sequences of the present invention comprise not only a nucleotide sequence selected from the group consisting of SEQ ID Nos:1-25 but also complementary sequences thereto. The complementary sequence includes a perfectly complementary sequence and substantially complementary sequence as well. The term “substantially complementary sequence” used herein means a nucleotide sequence hybridizable with a nucleotide sequence selected from the group consisting of SEQ ID Nos.1-25 under conventional stringent conditions. Details of “stringent conditions” are found in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Haymes, B. D., et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). The stringent conditions may be determined according to temperature, ionic strength (buffer concentration) and the presence of other compounds such as organic solvents. As understood by those of skill in the art, the stringent conditions are sequence dependent and are different under different environmental parameters. For example, the stringent conditions are provided by (i) washing with 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C. or (ii) hybridizing at 55° C. by use of, as a hybridization buffer, a solution containing 50% formamide, 2×SSC, and 10% dextran sulfate followed by highly stringent washing with 0.1×SSC containing EDTA at 55° C.
In addition, the nucleotide sequences of this invention comprise fragments of a nucleotide sequence selected from the group consisting of SEQ ID Nos:1-25.
It may be appreciated that numerous genes affect male germ cell development in birds and that some of these may be expressed in a testis-specific pattern. Accordingly, the testis-specific nucleotide sequences found in this invention could support such consensus in the art.
The novel nucleotide sequences of this invention can be applied to the following uses owing to their testis-specific expression pattern: (i) identification of testicular cells; (ii) discrimination of sex; (iii) separation of sperm; (iv) treatment or induction of infertility; and (v) gene diagnosis (specifically, gene diagnosis of infertility). For example, for discrimination of sex, total RNA extracted from early embryo is subjected to Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press), or cDNA prepared total RNA is subjected to hybridization reaction. Where the nucleotide sequences of this invention are applied to treatment of infertility, they may be introduced into testicular cells from birds having infertility due to gene deletion. In contrast, the nucleotide sequences of this invention contained in testicular cells of birds are manipulated to inhibit their expression for induction of infertility. Furthermore, the nucleotide sequences of this invention may be used for gene diagnosis, in particular, genetic polymorphism analysis.
In another aspect of this invention, there is provided a method for identifying a testicular cell of avian species, which comprises the steps of: (a) hybridizing a DNA molecule obtained from a cell with the testis-specific nucleotide sequence described above; and (b) analyzing the occurrence of the hybridization of step (a), wherein the occurrence of the hybridization is indicative of the testicular cell.
The testicular cell identified in this invention refers to a cell population present in testis tissue including spermatogonial stem cell, any germ cell (spermatogonial cell, sperm and spermatozoa) derived from spermatogonial stem cell, Sertoli cell, Leydig cell and muscle cell associated with connective tissue, preferably, spermatogonial stem cell, spermatogonial cell and sperm, more preferably, sperm, and most preferably, sexually mature sperm.
According to the method of this invention, it is preferred that the preparation of a DNA from cell to be analyzed is performed by reverse-transcribing mRNA isolated from the cell to obtain cDNA. In a specific example, RT-PCR (reverse transcriptase-PCR) is carried out to prepare cDNA. The isolation of total RNA may be performed according to conventional processes known in the art (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol. Biol. Rep., 9:242 (1991); Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Willey & Sons (1987); and Chomczynski, P. et al., Anal. Biochem. 162:156 (1987)). For instance, total RNA can be readily extracted from cells using TRIZOL (extraction solution).
The DNA (e.g. cDNA) prepared thus is preferably labeled. For labeling, materials detectable by spectroscopic measurement, photochemical measurement, biochemical measurement, bioelectronic measurement, immunochemical measurement, electronic measurement, chemical measurement are used. For instance, the labels include, but not limited to, radioisotopes such as P32 and S35, chemilluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers such as fluorescence markers and dyes, and magnetic labels. The dyes, for example, include, but not limited to, quinoline dye, triarylmethane dye, phthalein, azo dye and cyanine dye. The fluorescence makers include, but not limited to, fluorescein, phycoerythrin, rhodamine, lissamine, CY3 (cyanine fluorescent dye) and CY5 (cyanine fluorescent dye) (Pharmacia). Labeling is performed according to various methods known in the art, such as nick translation, random priming (Multiprime DNA labeling systems booklet, “Amersham” (1989)) and kination (Maxam & Gilbert, Methods in Enzymology, 65:499 (1986)).
According to the method of the present invention, the hybridization of a DNA obtained from a cell is carried out with referring to the procedures described in Southern, E. J. Mol. Biol. 98:503 (1975) in the hybridization conditions optimized through modifying several factors (salt concentration, temperature, reaction time and probe concentration) (Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)). For example, the hybridizing reaction may be performed by hybridizing at 55° C. by use of, as a hybridization buffer, a solution containing 50% formamide, 2×SSC, and 10% dextran sulfate followed by highly stringent washing with 0.1×SSC containing EDTA at 55° C.
According to the method of the present invention, the occurrence of hybridization is analyzed with various methods known in the art, particularly, depending on the types of labels used. For example, fluorescence microscope, preferably, confocal fluorescence microscope is used for fluorescence labels, and the intensity of the signal detected with such instruments increases proportionally to the extent of hybridization.
The present method permits to exactly identify testicular cells of avian species, preferably, a chicken, a quail, a turkey, a duck, a goose, a pheasant and a pigeon, more preferably, a chicken.
The following specific examples are intended to be illustrative of the invention and should not be construed as limiting the scope of the invention as defined by appended claims.
EXAMPLE I
Preparation of Tissues
The Korean native chickens (KNC) used in this study were kindly provided from the National Livestock Research Institute (NLRI) in Korea. The KNC had been domesticated in Korea approximately 2,000 years ago and KNC successfully adapted to local environments and climates. Although the KNC is considered to have relatively low capacity of egg and meat production compared to commercialized broiler or layer strains, the merits of KNC were also reported; the resistance to epidemic avian disease and the thick egg shell (Han S. W., et al., (1995) Estimation of Heritabilities and Genetic Correlations on Major Economic Traits in Korean Native Chicken. Korean Journal of Poultry Science 22, 67-75; and Ha J. K., et al., (1997) Studies on Thickness of Eggshell and Eggshell Membrane of Korean Native Chicken. Korean Journal of Poultry Science 24, 29-37).
In this study, brain, spleen, and testis tissues were dissected from brown-colored KNC at 26 weeks.
EXAMPLE II
Construction of CDNA Libraries and Sequencing
Samples of chicken tissues were homogenized and total RNA was isolated using TRIZOL (extraction solution) reagent (Invitrogen, Carlsbad, Calif., USA) and poly(A) mRNA was purified using the PolyATract mRNA isolation system (Promega, Wis., USA) according to the manufacturer's protocol. The libraries were synthesized using the ZAP-cDNA synthesis method (Stratagene, Calif., USA). The cDNA was prepared, size-fractionated, and inserted into the Uni ZAP XR vector using an XhoI linker-primer and EcoRI adaptor. After in vivo excision with E. coli strain SOLR, the cDNA libraries from the testis contained inserts ranging from 0.5 to 3 kb (n=20). After white/blue selection, colonies were picked randomly from rectangular plates (23×23 cm) and transferred to 384-well plates using a Q-BOT (robot system) (Genetix, UK). The plasmids were purified using a Montage Plasmid Miniprep 96 kit (Millipore, Mass., USA). Sequencing reactions were performed by priming at the 5′ end of cDNA, and analyzed on ABI 3700 automated DNA sequencers (PE Applied Biosystems, California, USA) using the manufacturer's protocols. We sequenced 17,502 cDNA clones from the libraries constructed from each tissue and could successfully read 13,132 EST sequences. FIG. 1 shows the proportion of the ESTs sequenced from each tissue.
EXAMPLE III
Bioinformatics Analysis
The chicken EST trace data were base-called using phrep program (Ewing B. & Green P. (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Research 8, 186-94) from trace chromatogram data of 3′-end EST sequences. The base-called EST data were vector-clipped, clustered, and assembled by using cross-match program and CAP3 program. (Huang X. & Madan A. (1999) CAP3: A DNA sequence assembly program. Genome Research 9, 868-77). Clustering and assembly of these ESTs resulted in total 6,957 unique assembled sequences with 1,699 contigs and 5,258 singletons and average number of ESTs per contig was about 4.7.
To identify putative novel transcripts, a sequence comparison between GGGI (Release 7.0) and our unique sequences were performed using stand-alone blast program (version 2.2.9) of the NCBI. The sequences with no blast hits were represented as the putative novel transcripts. In case of less than 300 bp in the sequenced length, we threw away for the fidelity of analysis. Total of 1,176 putative novel transcripts have resulted after the comparison and filtering, and mainly consisted of singletons (96%) in contrast with a small portion of contigs (4%). However, most of the sequences (93%) had no significant hits to the non-redundant (nr) protein databases of the NCBI in blastx search (e-value<0.00001). It might result from the low quality of sequences or sequencing errors. To filter out the low quality sequences, therefore, we aligned the EST sequences against chicken genome draft sequences of the University of California Santa Cruz (UCSC) genome browser. We used arbitrarily high degree of stringent condition for the transcript alignment with the genome sequences (score>500). About half of the sequences were screened out and finally resulted in 553 sequences (FIG. 2). As shown in FIG. 2, a large number (76%) of the assembled sequences that were tentatively considered as a novel transcript after aligned with the genome were mainly from testis-originated ESTs.
Furthermore, analysis of exon/intron structure of the contigs among the tentatively- and highly-qualified novel transcripts showed that the large portion of novel transcripts have intronless structure in coding regions. We used the Fisher's exact test to compare two independent binomial proportions between the EST contigs and others; the numbers of intronless contigs versus that of multiple exons contigs. Of the 37 contigs, 30 (81%) contigs have intronless structure in coding regions and this ratio of intronless contigs was too higher than 30% expected under the assumption of random sampling, in which one hundred TC sequences were randomly selected from the GGGI data for the analysis. The two-tailed P value was less than 0.0001 indicating extremely significant.
The large portion of single coding structure might be resulted from relatively short segment of the contigs or other systematic bias. However, many genes have been recently reported to be intronless and expressed exclusively in testicular germ cells (Hickox D. M., et al., (2002) Identification of a novel testis-specific member of the phosphatidylethanolamine binding protein family, pebp-2. Biology of Reproduction 67, 917-27; Miyagawa Y., et al., (2002) Molecular cloning and characterization of the human orthologue of male germ cell-specific actin capping protein alpha3 (cpalpha3). Molecular Human Reproduction 8, 531-9; Hisano M., et al., (2003) Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene, Tact1/Act17b, represses its expression in somatic cells. Nucleic Acids Research 31, 4797-804; Truong A., et al., (2003) Isolation and expression analysis of the canine insulin-like factor 3 gene. Biology of Reproduction 69, 1658-64; Hao Z., et al., (2004) Expression analysis of the human testis-specific serine/threonine kinase (TSSK) homologues. A TSSK member is present in the equatorial segment of human sperm. Molecular Human Reproduction 10, 433-44; Choi I., et al., (2004) Characterization and comparative genomic analysis of intronless Adams with testicular gene expression. Genomics 83, 636-46). Some of these genes have functionality and the others may be non-functional pseudo-genes. One proposed mechanism of intron loss is reverse transcription of a proceesed mRNA followed by gene conversion. Betran et al. (Retroposed new genes out of the X in Drosophila. Genome Research, 12:1854-9 (2002)) suggested that these retrogenes showed a significant excess of retrogenes that originated from the X chromosome in Drosophila. Furthermore, most of the X-derived autosomal retrogenes had evolved a testis expression pattern.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.

## Claims

1. A method for identifying a testicular cell of a chicken, which comprises the steps of:
(a) isolating mRNA from a cell of a chicken,
(b) reverse transcribing the mRNA to obtain cDNA,
(c) hybridizing the cDNA with the nucleotide sequence of SEQ ID NO: 7, and
(d) analyzing the occurrence of the hybridization of step (c), wherein the occurrence of the hybridization is indicative of a testicular cell.