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Preparing and Analyzing Expressed Sequence Tags (ESTs) Library for the Mammary Tissue of Local Turkish Kivircik Sheep.

1. Introduction

Turkey is an important country in sheep husbandry and there are 33.2 million sheep in the country [1]. Kivircik sheep spread to the Thrace region, Marmara region, and the North Aegean region. They are fed for quality meat, milk, and wool. They are adapted to adverse environmental conditions and resistant parasites. Birth and adult body weight and daily weight gain are 3.7-4 kg, 50-70 kg, and 263 g, respectively. In addition, lactation period is 180 days, lactation milk yield is 83 kg, and wool production yield is 1.5 kg [2].

Colostrum is the first lacteal secretion from the mammary tissue of the mammals after birth [3]. Compared to normal milk, colostrum is clearly richer in contents of immunoglobulin, growth factors, protein, nonprotein nitrogen, fat, ash, minerals, and vitamins [4]. Chemical composition and immunoglobulin level particularly in colostrums exhibits a change within the first 24 h after birth [5-7]. It is only secreted during the first 72 hours of lactation [8] and the excretion is intense at the first 12-36 hours [7]. Sufficient colostrums intake of newborn lambs in the first days of their lives plays essential role in their healthy growth and reaching to ideal market weight [9]. The raisers are desired to breed very productive animal, so colostrums secretion is a crucially important stage for breeding. Because the compounds of colostrums are important, the genes which are related to colostrums secretion have been the center of interest.

A cDNA library containing the information from the mRNA of a particular tissue or organism is an efficient tool for research on gene structure, function, and manipulation [10]. The production of Expressed Sequence Tags (ESTs) begins with the construction of cDNA libraries. The first description of EST was reported from humans in 1991 [11]. ESTs, which are obtained in the results of sequencing of cDNA clones, are very important data for genomics studies [12].

In our study, we used one of the native breeds known as Kivircik sheep in Turkey. First of all, mammary tissues of Kivircik sheep were collected in two stages, before parturition and after parturition (the secretion of colostrums known to be considerably high), and two different cDNA libraries were constructed from those tissues. By using the bioinformatic tools, the ESTs were analyzed. Finally, obtained ESTs were compared with other genes of distinct organisms found in databases and putative functions of the genes were estimated. We aimed in this study to obtain mammary gland of gene expression profile at prenatal and postnatal stages and to compare the genetic components of colostrums secretion according to these two stages in Kivircik sheep.

2. Materials and Methods

2.1. Tissue Material. Kivircik sheep (Ovis aries) in farm of the Faculty of Veterinary Science at University of Istanbul was used in this study. The mammary tissues from the same sheep were taken by biopsy in 6-8 h during high period of the colostrums secretion after parturition and one week before parturition.

2.2. cDNA Library Construction, Quality Controls, and Sequencing. Total RNA was isolated from 0,377 gr prenatal stage and 1,316 gr postnatal stage mammary tissues with the RNeasy Kit (Qiagen). mRNA was made pure from total RNA using the Oligotex Spin-Column Protocol (Oligotex mRNA Mini Kit, Qiagen, Valencia, CA). Two different cDNA libraries for pre- and postnatal stage tissue were established with 0,23 [micro]g and 0,75 [micro]g mRNA, respectively. cDNA libraries were constructed with the CloneMiner cDNA Library Construction Kit according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Double-stranded cDNA was cloned into pDONR222 vector and transformed into E. coli strain DH5 (Invitrogen, Carlsbad, CA, USA). Each cDNA library was plated onto LB-kanamycin agar medium and individual grown colonies were picked into 384-well plates with SOB medium and inoculated overnight. After the addition of glycerol (10% v/v), the library was stored at -80[degrees]C.

Plasmid DNA was isolated from casually selected 142 clones with alkaline lysis method [13]. Isolated DNA was digested with Bgl1701 and analyzed by 1% agarose gel electrophoresis for identifying insert size.

As a template, randomly selected 3072 clones were used for PCR amplification of the cloned cDNA by M13 universal primers. Automated sequencing was performed on an automated high-throughput pipeline using the ABI 3730 capillary sequencer (PE Applied Biosystems, Foster City, CA) at the Genome Sequencing Center, Washington University in St. Louis (WUSTL).

2.3. Sequence Analysis. For analysis of the raw EST data, the low-quality, adapter, and the vector sequences were removed with Phred software [14, 15] (CodonCode Corp., Dedham, MA). The remaining EST sequences were reprocessed by using "cross-match" program which is application of Phrap for the vector sequence trimming [14, 15].

Prenatal and postnatal period EST sequences were assembled separately into contigs with Contig Assembly Program 3 (CAP3) [16, 17]. The default values were used for all the parameters. Results were evaluated by using BEAP program which was developed for editing and representing of alignment [18]. Putative functions of all unique sequences and contigs were designated by gene homology based on BLAST [19]. For Blastn and statistical analysis of all the EST sequences, Geneious software was used [20]. ESTs that showed high similarity in GenBank were classified according to molecular function, biological process, cellular component, protein class, and pathway, respectively, based on Panther classification [21].

3. Results

3.1. Characterization of cDNA Libraries. Two different cDNA libraries were constructed at prenatal and postnatal stages from mammary gland tissue. In cDNA library for the prenatal period, there occurred 3.6 x [10.sup.3] clones and its average insert length was 1 kb (min 200 bp to 1500 bp) (Figure 1). On the other hand, cDNA library for postnatal stage, which consisted of 4.3 x [10.sup.3] clones, has an average insert length of 0.9 kb ranging from 200 to 1500 kb (Figure 2). After construction of cDNA libraries, 1536 clones were randomly selected and sequenced from prenatal stage library; 1536 clones were randomly selected and sequenced from postnatal stage library. Therefore, 3072 EST sequences were generated.

3.2. BLAST Analysis of ESTs. From obtained 3072 EST sequences, raw EST data were processed and base-called with Phred-Cross match computer program. The EST sequences were trimmed and vector, adapter, and low-quality bases removed. While 318 low-quality sequences were determined in prenatal cDNA library, 340 low-quality sequences were removed in cDNA library which was constructed at postnatal stage (Table 1). The remaining 1218 high-quality prenatal EST sequences and 1196 high-quality postnatal EST sequences were achieved. Putative functions of all unique sequences were designated by gene homology based on BLAST. Blastn analysis was done using Geneious software. Obtained data was evaluated statistically by this program.

According to these results, length of prenatal ESTs changes between 542 bp and 1587 bp; the shortest postnatal EST and the longest postnatal EST were 605 bp and 1535 bp, respectively (Table 1 and Figures 3 and 4).

Pursuant to Blastn results of 1218 prenatal ESTs, 154 of them (12.7%) which showed [greater than or equal to] 80 similarity or e value [less than or equal to] 1e - 10 to known sequences of other organisms in NCBI were determined. Also the 1001 ESTs (82.3%) showed significant similarities to putative genes with score of below 80% homology and 63 ESTs had no similarity to situated sequences in databases.

Among the 1196 postnatal ESTs, 268 of them (22.4%) showed 80% and higher similarity; 923 ESTs (77.2%) showed similarities with score of <80 bits. Also 38 ESTs did not match any registrated sequences in NCBI.

All 2414 EST sequences were submitted to GenBank. ESTs were registered to NCBI under the accession numbers of GW996847 to GW999260.

3.3. GO Analysis of ESTs. Total 422 ESTs which show 80% and over 80% similarity to known sequences of other organisms in NCBI were classified according to GO terms such as molecular function, biological process, cellular components, protein class, and pathway with PANTHER database (Figures 5 and 6). 154 and 268 ESTs of these total ESTs were prenatal and postnatal ESTs, respectively. According to the statistical results of PANTHER of high similarity prenatal and postnatal ESTs, 137 and 127 different putative genes were listed and classified, respectively, by GO terms. 39 prenatal ESTs and 37 postnatal EST were uncategorized.

9 different types of molecular functions were found in prenatal ESTs. The common molecular function GO terms are "binding" which consists of 43 proteins and "catalytic activity" which consists of 27 proteins. Moreover, most of the postnatal ESTs in the molecular function GO term were assigned the same categories, "binding" and "catalytic activity." In the biological process category that recognizes series of events or molecular functions, 56 and 30 of prenatal ESTs and 55 and 20 of postnatal ESTs were viewed in "metabolic process" and "cellular process," respectively. The cellular component GO identifies locations at the levels of subcellular structures and macromolecular complexes. In this GO term category, the prenatal ESTs were accumulated more than postnatal ESTs in "cell part," "macromolecular complex," and "organelle." According to protein class GO term classification of Panther database, 23 of prenatal ESTs and 20 of postnatal ESTs most observed "nucleic acid binding" category. On the other hand, Panther database gives information about pathway GO term. When we analyzed prenatal and postnatal ESTs, "B cell activation," "T cell activation," and "Huntington disease" categories included some prenatal proteins; moreover, "apoptosis signaling pathway," "p53 pathway," and "Parkinson disease" categories contained some postnatal proteins.

3.4. Analyzing of Contigs. 1218 high-quality prenatal EST sequences and 1196 high-quality postnatal EST sequences were appointed to achieve contig. Fragment assembly was done with the CAP3 software. According to CAP3 result the prenatal stage EST sequences into 23 contigs and postnatal stage EST sequences into 27 contigs were assembled. Furthermore, in prenatal stage contigs, the longest contig is 1394 bp, it is 2068 in postnatal stage contig. The number of singlets is 1164 and 1059, respectively (Table 2).

The results which were obtained by CAP3 software were evaluated with the program BEAP which was developed for editing and representing of alignment (Figure 7).

All of the contigs were designated by Blastn in NCBI. The Blastn results of prenatal and postnatal contigs are shown in Tables 3 and 4, respectively.

Among the Blastn results of prenatal contigs, 7 of them showed high similarities. These are a unique protein associated with intracellular transfer of membrane by coated vesicles "clathrin," protein that plays role in protein synthesis, "elongation factor-1," a member of the mitochondrial carrier family, "alpha solute carrier family, member 13," protein that related with immune system, "immunoglobulin gamma one chain," a gene that encodes the glycodelin protein, "progestagen-associated endometrial protein (PAEP)," a major component of a specific type of lipoprotein called very low-density lipoproteins (VLDLs), and "apolipoprotein E." Additionally, 10 of 23 prenatal contigs showed similarities to putative different ribosomal proteins in NCBI with score of <80 (Table 3).

As shown in Table 4, 11 of 27 postnatal contigs have similarities with score of [greater than or equal to]80 bits or e value [less than or equal to] [10.sup.-10] according to Blastn. 3 postnatal contigs of high similarity postnatal contigs include 3 different ribosomal proteins. In addition, 5 postnatal contigs interacte with calcium and calcium phosphate and self-aggregate to be organized into a supramolecular structure "alpha casein S1 and S2." The other two contigs show high similarities to putative proteins which are "sperm associated antigen 8 (SPAG8)" and "translation elongation factor 1 alpha." Important putative genes of the postnatal contigs which showed resemblance below 79% in NCBI are "collagen type III alpha 1," "nuclear protein, transcriptional regulator," "beta-2-microglobulin," "thymosin beta 4," "beta-casein," and different ribosomal proteins.

4. Discussion

EST projects are powerful tools for analyzing gene expression patterns in a given tissue and/or at a certain stage and the identification of genes [22-24]. These technologies are very important for obtaining genomic sequence information of organisms [25]. In the molecular studies, generation of ESTs is a perfect and unique approach because it allows both expression and estimation and discovery of new genes to be conducted at the same time. Consequently, analysis of expression of a large number of gene profile supplies for scientists to find their functions and facilitates the understanding activity of biological processes in specific tissue or cell of organisms at the certain stage [26].

In this study, we obtained two different EST libraries from Kivircik sheep mammary gland at one week before parturition and 6-8 h after parturition. Because colostrums are only secreted during the first 72 hours after parturition [8], we aimed in this project to achieve putative gene profiling at different time and find the genetic components of colostrums secretion in Kivircik sheep.

The results of the analyzing contig and GO analysis of ESTs and the Blastn analysis of the ESTs showed 80% and higher similarity to putative genes in NCBI. Moreover, we have determined gene expression profile of mammary gland at the different developmental stages, which are prenatal and postnatal.

Table 5 shows Blastn results of some breed-specific expressed genes in all ESTs of prenatal and postnatal ESTs. According to these results, levels of ribosomal protein, transcription factors, and translation factors were found about the same level at both stages. CREB2F, SRF, AKNA, CRTC2, TCF20, and SPDEF transcription factors were observed in prenatal ESTs and also ELF5, NUPR1, BTAF1, TCF12, TFE3, and TCF20 transcription factors were observed in postnatal ESTs. Due to the high level of protein synthesis in the lactation phase, expression of the proteins that participate in ribosomal structure increases at the preparation lactation stage. Also, the genes that have function in transcriptional and translational regulation are expressed highly before parturition [27]. Additionally, we have observed known putative genes related to immune system, growth, and lipid metabolism. These expressed genes which improve quality of milk are "immunoglobulins," "MHC," and "beta-2-microglobulin." Because lactation begins few weeks before parturition [28] and we choose sequenced cDNA clones randomly, these putative genes are found approximately at the same rate in prenatal and postnatal ESTs. Besides, expressions of genes related to milk production are minimal, because the milk is not secreted from mammary gland before the parturition. On the other hand, the milk proteins that affect milk quality are defined as significant milk genes. We have observed their expressions to be high in the lactation stage such as "caseins (alpha-S1, alpha-S2, beta, and kappa casein)" and whey proteins "beta-lactoglobulin" and "alpha-lactalbumin." Expression of the milk proteins was slightly near the end of gestation, which can be used as a symbol of the maturity of mammary epithelial cells [29].

On the other hand, in respect of GO analysis, GO terms were identified and observed on five categories, such as molecular function, biological process, cellular components, protein class, and pathways. Prenatal and postnatal high-similarity ESTs were classified with Panther database according to GO terms. According to comparison of these results, prenatal and postnatal ESTs were classified almost in the same category.

After our study, using large-scale EST sequencing as strategy, we have constructed Kivircik sheep mammary gland gene expression profiles in different stages and found prelactation and lactation (especially secreting colostrum) specific genes. These results will help us understand the comparison of mammary gland gene expression profiles between two distinct stages and provide new clues for genomic research. Because our ESTs data just only matched the other organisms in NCBI by approximately 50%, it will also provide the increasing number of ESTs of Ovis aries in NCBI Genbank database and new genes about Ovis aries. This work will improve with the research of different developmental stage of mammary gland.

http://dx.doi.org/10.1155/2017/9604762

Competing Interests

The authors declare that there are no competing interests regarding the publication of this paper.

Acknowledgments

This research has been supported by Yildiz Technical University Scientific Research Projects Coordination Department (Project no. 27-01-07-02).

References

[1] TurkStat, Animal Production Statistics, 2016, http://www.tuik .gov.tr/.

[2] General Directorate of Agricultural Research and Policie, Turkiye Evcil Hayvan Genetik Kaynaklari Tanitim Katalogu, General Directorate of Agricultural Research and Policie, Ankara, Turkey, 2009.

[3] R. Pakkanen and J. Aalto, "Growth factors and antimicrobial factors of bovine colostrum," International Dairy Journal, vol. 7, no. 5, pp. 285-297, 1997.

[4] F. O. Uruakpa, M. A. H. Ismond, and E. N. T. Akobundu, "Colostrum and its benefits: a review," Nutrition Research, vol. 22, no. 6, pp. 755-767, 2002.

[5] S. E. Pattinson and E. W. Thomas, "The effect of sire breed on colostrum production of crossbred ewes," Livestock Production Science, vol. 86, no. 1-3, pp. 47-53, 2004.

[6] A. Arguello, N. Castro, M. J. Zamorano, A. Castroalonso, and J. Capote, "Passive transfer of immunity in kid goats fed refrigerated and frozen goat colostrum and commercial sheep colostrum," Small Ruminant Research, vol. 54, no. 3, pp. 237-241, 2004.

[7] J. C. Chen, C. J. Chang, H. C. Peh, and S. Y. Chen, "Serum protein levels and neonatal growth rate of Nubian goat kids in Taiwan area," Small Ruminant Research, vol. 32, no. 2, pp. 153-160, 1999.

[8] S. Kracmar, J. Kuchtik, M. Baran et al., "Dynamics of changes in contents of organic and inorganic substances in sheep colostrum within the first 72 h after parturition," Small Ruminant Research, vol. 56, no. 1-3, pp. 183-188, 2005.

[9] H. S. Umberger, "Profitable artificial rearing of lambs," Animal and Poultry Sciences Publication No. 410-023, Virginia Cooperative Extension, 1997.

[10] S.-Y. Ying, "Complementary DNA libraries: an overview," Applied Biochemistry and Biotechnology--Part B Molecular Biotechnology, vol. 27, no. 3, pp. 245-252, 2004.

[11] M. D. Adams, J. M. Kelley, J. D. Gocayne et al., "Complementary DNA sequencing: expressed sequence tags and human genome project," Science, vol. 252, no. 5013, pp. 1651-1656, 1991.

[12] L.-X. Du, S.-F. Liu, J. Zhu et al., "Construction of SMART cDNA library of sheep ovary and identification of candidate gene by homologous cloning," Agricultural Sciences in China, vol. 6, no. 11, pp. 1390-1395, 2007.

[13] J. Sambrook, E. F. Fritsch, and T. Maniatis, Moleculer Clonning: A Laboratory Manuel, vol. 2nd, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA, 1989.

[14] B. Ewing, L. Hillier, M. C. Wendl, and P. Green, "Base-calling of automated sequencer traces using phred. I. accuracy assessment," Genome Research, vol. 8, no. 3, pp. 175-185, 1998.

[15] B. Ewing and P. Green, "Base-calling of automated sequencer traces using phred. II. Error probabilities," Genome Research, vol. 8, no. 3, pp. 186-194, 1998.

[16] X. Huang and A. Madan, "CAP3: a DNA sequence assembly program," Genome Research, vol. 9, pp. 868-877, 1999.

[17] X. Huang, "An improved sequence assembly program," Genomics, vol. 33, no. 1, pp. 21-31, 1996.

[18] J. E. Koltes, Z.-L. Hu, E. Fritz, and J. M. Reecy, "BEAP: the BLAST extension and alignment program--a tool for contig construction and analysis of preliminary genome sequence," BMC Research Notes, vol. 2, article 11, 2009.

[19] S. F. Altschul, W. Gish, W. Miller, E.W. Meyers, and D. J. Lipman, "Basic local alignment search tool," Journal of Molecular Biology, vol. 215, no. 3, pp. 403-410, 1990.

[20] M. Kearse, R. Moir, A. Wilson et al., "Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data," Bioinformatics, vol. 28, no. 12, pp. 1647-1649, 2012.

[21] P. D. Thomas, M. J. Campbell, A. Kejariwal et al., "PANTHER: a library of protein families and subfamilies indexed by function," Genome Research, vol. 13, no. 9, pp. 2129-2141, 2003.

[22] S. Chen, H. Miao, F. Chen, B. Jiang, J. Lu, and W. Fang, "Analysis of expressed sequence tags (ESTs) collected from the inflorescence of chrysanthemum," Plant Molecular Biology Reporter, vol. 27, no. 4, pp. 503-510, 2009.

[23] W.-K. Wang, C.-C. Liu, T.-Y. Chiang, M.-T. Chen, C.-H. Chou, and C.-H. Yeh, "Characterization of expressed sequence tags from flower buds of alpine Lilium formosanum using a subtractive cDNA library," Plant Molecular Biology Reporter, vol. 29, no. 1, pp. 88-97, 2011.

[24] D. Yang, Z. H. Tang, L. P. Zhang, C. P. Zhao, and Y. L. Zheng, "Construction, characterization, and expressed sequence tag (EST) analysis of normalized cDNA library of thermo-photoperiod-sensitive genic male sterile (TPGMS) wheat from spike developmental stages," Plant Molecular Biology Reporter, vol. 27, no. 2, pp. 117-125, 2009.

[25] Y. Gueguen, J.-P. Cadoret, D. Flament et al., "Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostreagigas," Gene, vol. 303, pp. 139-145, 2003.

[26] T. Thanh, V. T. Q. Chi, M. P. Abdullah et al., "Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda," Molecular Biology Reports, vol. 38, no. 1, pp. 177-182, 2011.

[27] Z. Su, X. Dong, B. Zhang et al., "Gene expression profiling in porcine mammary gland during lactation and identification of breed-and developmental-stage-specific genes," Science in China Series C, vol. 49, no. 1, pp. 26-36, 2006.

[28] Y. Gao, X. Lin, K. Shi, Z. Yan, and Z. Wang, "Bovine mammary gene expression profiling during the onset of lactation," PLoS ONE, vol. 8, no. 8, Article ID e70393, 2013.

[29] G. W. Robinson, R. A. McKnight, G. H. Smith, and L. Hennighausen, "Mammary epithelial cells undergo secretory differentiation in cycling virgins but require pregnancy for the establishment of terminal differentiation," Development, vol. 121, no. 7, pp. 2079-2090, 1995.

Nehir Ozdemir Ozgenturk, (1) Zehra Omeroglu Ulu, (1) Salih Ulu, (1) Cemal Un, (2) Kemal Ozdem Oztabak, (3) and Kemal Altunatmaz (4)

(1) Faculty of Art and Science, Molecular Biology and Genetics, Yildiz Technical University, Istanbul, Turkey

(2) Department of Biology, Faculty of Art and Science, Ege University, Izmir, Turkey

(3) Department of Biochemistry, Faculty of Veterinary Medicine, Istanbul University, Istanbul, Turkey

(4) Department of Surgery, Faculty of Veterinary Medicine, Istanbul University, Istanbul, Turkey

Correspondence should be addressed to Nehir Ozdemir Ozgenturk; nehirozdemir@yahoo.com

Received 18 October 2016; Accepted 7 December 2016; Published 23 January 2017

Academic Editor: Ferenc Olasz

Caption: Figure 1: (a) Prenatal cDNA clones obtaining with alkaline lysis method. (b) Cutting of prenatal cDNA clones with Bsp1407I restriction enzyme.

Caption: Figure 2: (a) Postnatal cDNA clones obtaining with alkaline lysis method. (b) Cutting of postnatal cDNA clones with Bsp1407I restriction enzyme.

Caption: Figure 3: The length of prenatal ESTs.

Caption: Figure 4: The length of postnatal ESTs.

Caption: Figure 7: The result of Contig 20 that was obtained by CAP3 with BEAP.
Table 1: The results of analyzing ESTs with Phred software and
Geneious software.

                         Prenatal period   Postnatal period

EST number                    1536               1536
Low quality EST number         318               340
Quality EST number            1218               1196
Average of ESTs (bp)          1023               1026
The shortest EST (bp)          542               605
The longest EST (bp)          1587               1535

Table 2: The analysis of prenatal and postnatal contigs by CAP3.

                          Prenatal period   Postnatal period

Contig number                   23                 27
Singlet number                 1164               1059
The longest contig (bp)        1394               2068
The shortest contig (bp)        765               994

Table 3: The Blastn results of prenatal contigs.

Contig                                   Query      Max.
number             Description          coverage   ident.

Contig 1      Bos taurus cDNA clone       56%       92%
                 IMAGE: 7944277

              Bos taurus ribosomal
                protein S25, mRNA
Contig 2        (cDNA clone MGC:          45%       92%
                 127761, IMAGE:
             7962930), complete cds

             Homo sapiens BAC clone
Contig 3       RP11-67M24 from 4,         59%       72%
                complete sequence

               Bos taurus thymosin
             beta 4, X-linked, mRNA
Contig 4        (cDNA clone MGC:          73%       94%
                 157367, IMAGE:
             8260878), complete cds

            Equus caballus clathrin,
                light chain (Lca)
                 (CLTA), mRNA >
Contig 5      dbj|AB495091.1| Equus       85%       96%
              caballus clc mRNA for
              clathrin light chain,
                  complete cds

              PREDICTED: Ovis aries
Contig 6    elongation factor-1 alpha     83%       98%
              (LOC100125610), mRNA

              Ovis aries ribosomal
Contig 7      protein S19 (RPS19),        47%       98%
                      mRNA

              PREDICTED: Ovis aries
            solute carrier family 25
Contig 8      (aspartate/glutamate        80%       96%
               carrier), member 13
                (SLC25A13), mRNA

                O. aries mRNA for
Contig 9         immunoglobulin           88%       87%
             gamma-1 chain secreted
                      form

                   PREDICTED:
            Pantholops hodgsonii 60S
Contig 10       ribosomal protein         62%       93%
                    L32-like
              (LOC102314866), mRNA

              Bos taurus ribosomal
              protein L31 (RPL31),
              mRNA > gb|BC102125.1|
Contig 11     Bos taurus ribosomal        47%       92%
                protein L31, mRNA
                (cDNA clone MGC:
                 127011, IMAGE:
             7942112), complete cds

              Bos taurus ribosomal
             protein L13a (RPL13A),
              mRNA > gb|BC103039.1|
              Bos taurus ribosomal
Contig 12      protein L13a, mRNA         54%       95%
                (cDNA clone MGC:
                 128198, IMAGE:
               7984430), complete
                       cds

              Bos taurus ribosomal
               protein S7 (RPS7),
              mRNA > gb|BC146133.1|
Contig 13     Bos taurus ribosomal        68%       93%
                protein S7, mRNA
                (cDNA clone MGC:
                 165697, IMAGE:
             8068046), complete cds

               Capra hircus serum
Contig 14          amyloid A3             67%       92%
              (LOC100860781), mRNA

             Capra hircus ribosomal
Contig 15      protein S18 (RPS18)        73%       95%
                 mRNA, complete
                       cds

                   Bos taurus
Contig 16    progestagen-associated       84%       92%
               endometrial protein
                  (PAEP), mRNA

              PREDICTED: Ovis aries
Contig 17     ribosomal protein S11       49%       94%
                 (LOC100037666),
                      mRNA

             Capra hircus ribosomal
Contig 18      protein S18 (RPS18)        40%       97%
                 mRNA, complete
                       cds

             Bos taurus endothelial
             differentiation-related
              factor 1(EDF1), mRNA
              > gb|BC102246.1| Bos
Contig 19      taurus endothelial         74%       88%
             differentiation-related
              factor 1, mRNA (cDNA
               clone MGC: 127033,
                IMAGE: 7942128),
                  complete cds

               PREDICTED: Bubalus
             bubalis uncharacterized
Contig 20         LOC102396380            64%       83%
                 (LOC102396380),
                    misc.RNA

              Bos taurus ribosomal
              protein L11 (RPL11),
              mRNA > gb|BC102524.1|
              Bos taurus ribosomal
Contig 21       protein L11, mRNA         63%       95%
                (cDNA clone MGC:
                 127967, IMAGE:
             7956291), complete cds

              Bos taurus ribosomal
Contig 22     protein L7a (RPL7A),        46%       87%
                      mRNA

                   Bos taurus
             apolipoprotein E, mRNA
Contig 23       (cDNA clone MGC:          87%       90%
                 127905, IMAGE:
             7962554), complete cds

Contig       Length of      The number of
number      contig (bp)   ESTs in the contig

Contig 1        768               4

Contig 2       1121               2

Contig 3       1049               2

Contig 4        860               2

Contig 5       1193               2

Contig 6       1083               5

Contig 7       1171               4

Contig 8       1049               2

Contig 9       1394               2

Contig 10       842               2

Contig 11      1088               2

Contig 12      1239               2

Contig 13      1036               2

Contig 14       875               4

Contig 15       765               3

Contig 16      1032               4

Contig 17      1101               2

Contig 18      1297               2

Contig 19      1082               2

Contig 20       890               2

Contig 21       965               2

Contig 22      1097               2

Contig 23      1288               3

Table 4: The Blastn results of postnatal contigs.

Contig                                  Query      Max.
number            Description          coverage   ident.

                PREDICTED: Capra
                  hircus sperm
Contig 1      associated antigen 8       87%       90%
              (SPAG8), transcript
              variant X3, misc_RNA

                PREDICTED: Capra
              hircus collagen type
Contig 2          III alpha 1            67%       87%
                (LOC100860938),
                      mRNA

                PREDICTED: Capra
            hircus nuclear protein,
Contig 3        transcriptional          58%       95%
             regulator, 1 (NUPR1),
                      mRNA

             Bos taurus eukaryotic
             translation elongation
               factor 1 alpha 1,
Contig 4        mRNA (cDNA clone         95%       90%
                  MGC: 126913,
                IMAGE: 7928985),
                  complete cds

              Bos taurus ribosomal
               protein S3 (RPS3),
                     mRNA >
               gb|BC102090.1| Bos
Contig 5        taurus ribosomal         82%       92%
                protein S3, mRNA
                (cDNA clone MGC:
                 127206, IMAGE:
               7945675), complete
                      cds

              Solanum lycopersicum
Contig 6        chromosome ch12,         30%       67%
                complete genome

                   PREDICTED:
              Pantholops hodgsonii
               GTPase IMAP family
Contig 7         member 5-like           17%       91%
                (LOC102329836),
             transcript variant X5,
                      mRNA

Contig 8       C. hircus mRNA for        83%       91%
                   as1-casein

                   Bos taurus
              beta-2-microglobulin
                 (B2M), mRNA >
              gb | BC118352.11 Bos
Contig 9             taurus              79%       89%
             beta-2-microglobulin,
                mRNA (cDNA clone
                  MGC: 140690,
                IMAGE: 8275195),
                  complete cds

                   Ovis aries
                alpha-S1-casein
Contig 10       (csn1s1), mRNA >         83%       95%
              emb|X03237.1| Sheep
                    mRNA for
                alpha-S1-casein

                   Ovis aries
                alpha-S2-casein
Contig 11      (LOC443383), mRNA         85%       91%
                > emb|X03238.1|
                 Sheep mRNA for
                alpha-S2-casein

                   Ovis aries
                alpha-S2-casein
Contig 12      (LOC443383), mRNA         82%       95%
                > emb|X03238.1|
                 Sheep mRNA for
                alpha-S2-casein

             Ovis aries breed Small
                 Tail Han Sheep
Contig 13       thymosin beta 4,         60%       95%
               X-linked (ThymB4X)
               mRNA, complete cds

Contig 14        Sheep mRNA for          79%       90%
                  beta-casein

                 PREDICTED: Bos
                     taurus
Contig 15      N-ethylmaleimide-         80%       93%
               sensitive factor,
              transcript variant 4
                  (NSF), mRNA

              Bos taurus ribosomal
              protein S4, X-linked
                (RPS4X), mRNA >
              gb | BC102476.11 Bos
Contig 16       taurus ribosomal         84%       92%
            protein S4, Y-linked 2,
                mRNA (cDNA clone
                  MGC: 127573,
                IMAGE: 7950762),
                  complete cds

              Bos taurus ribosomal
               protein L27a, mRNA
Contig 17       (cDNA clone MGC:         38%       93%
                 133578, IMAGE:
               8058503), complete
                      cds

                     Bovine
Contig 18     alpha-S2-casein type       66%       89%
               A protein (CASAS2)
                gene, exons 1-18

                   Ovis aries
                alpha-S1-casein
Contig 19       (csn1s1), mRNA >         85%       93%
              emb|X03237.1| Sheep
                    mRNA for
                alpha-S1-casein

               Ovis aries casein
                 kappa (CSN3),
Contig 20            mRNA >              79%       95%
              emb|X51822.1| Sheep
               kappa-Cn mRNA for
                  kappa-casein

                PREDICTED: Capra
              hircus 40S ribosomal
Contig 21       protein S15-like         45%       96%
                (LOC102191702),
                      mRNA

               Ovis aries clone 7
Contig 22     ribosomal protein SA       50%       97%
               (RPSA) pseudogene,
               complete sequence

              Bos taurus ribosomal
               protein S3A, mRNA
Contig 23       (cDNA clone MGC:         86%       91%
                 127088, IMAGE:
               7943245), complete
                      cds

                PREDICTED: Ovis
Contig 24       aries ribosomal          75%       90%
               protein L6 (RPL6),
                      mRNA

                   PREDICTED:
              Pantholops hodgsonii
Contig 25    40S ribosomal protein       45%       96%
                    S25-like
                (LOC102318811),
                      mRNA

                Bubalus bubalis
Contig 26       alpha-S1-casein          82%       93%
               mRNA, complete cds

              Ovis aries cell line                 97%
Contig 27     1LL2 mitochondrion,        79%
                complete genome

Contig       Length of    The number of
number      contig (bp)   ESTs in contig

Contig 1       1015             2

Contig 2       1332             2

Contig 3       1039             3

Contig 4       1992             10

Contig 5       1074             2

Contig 6       2068             2

Contig 7       1031             2

Contig 8       1072             3

Contig 9       1383             2

Contig 10      1283             5

Contig 11      1042             4

Contig 12      1071             13

Contig 13      1048             2

Contig 14      1487             21

Contig 15      1116             2

Contig 16      1088             2

Contig 17      1361             2

Contig 18      1054             2

Contig 19       994             2

Contig 20      1105             3

Contig 21      1060             2

Contig 22      1318             2

Contig 23      1069             2

Contig 24      1150             2

Contig 25      1079             2

Contig 26      1439             38

Contig 27      1059             3

Table 5: The percentage and number of some expressed genes
in prenatal and postnatal ESTs.

                         Prenatal ESTs
Genes                         EST%        Number of
                                            ESTs

Casein (alpha-S1,             1,2%           15
alpha-S2, beta, kappa)
Immunoglobulins               2,9%           36
MHC                           1,6%           20
Translation factors,          2,3%           29
elongation factors
Transcription factors
(CREB2F, SRF, AKNA,           0,4%            6
CRTC2, TCF20, SPDEF)
Ribosomal proteins           15,1%           184
Growth factors                0,3%            4
Beta-lactoglobulin            2,2%           27
Lactalbumin                    0              0
Beta-2-microglobulin          0,4%            5
Fatty acid synthase           0,5%            7
(FASN)
Fatty acid binding             0              0
protein

                         Postnatal ESTs   Number of
Genes                         EST%          ESTs

Casein (alpha-S1,            23,1%           277
alpha-S2, beta, kappa)
Immunoglobulins               1,4%           17
MHC                           0,4%            5
Translation factors,          2,5%           31
elongation factors
Transcription factors
(ELF5, NUPR1, BTAF1,          0,6%            8
TCF12, TFE3, TCF2)
Ribosomal proteins           14,5%           174
Growth factors                0,2%            3
Beta-lactoglobulin            1,9%           23
Lactalbumin                   0,6%            8
Beta-2-microglobulin          0,4%            6
Fatty acid synthase          0,08%            1
(FASN)
Fatty acid binding            0,3%            4
protein

Figure 5: GO terms distribution in the molecular function (a), the
biological process (b), the cellular components (c), and the protein
class d) for prenatal ESTs.

(a)

                                                              Molecular
                                                              functions

Transporter activity (GO:0005215)                                 2
Translation regulator activity (GO:0045182)                       2
Protein binding transcription factor activity (GO:0000988)        1
Enzyme regulator activity (GO:0030234)                            6
Catalytic activity (GO:0003824)                                  27
Receptor activity (GO:0004872)                                    6
Nucleic acid binding transcription factor activity                7
(GO:0001071)
Structural molecule activity (GO:0005198)                        16
Binding (GO:0005488)                                             43

(b)

                                                             Biological
                                                               process

Cellular component organization or biogenesis (GO:0071840)        8
Cellular process (GO:0009987)                                    30
Localization (GO:0051179)                                        14
Apoptotic process (GO:0006915)                                    5
Reproduction (GO:0000003)                                         3
Biological regulation (GO:0065007)                               17
Response to stimulus (GO:0050896)                                 8
Developmental process (GO:0032502)                               11
Multicellular organismal process (GO:0032501)                     6
Biological adhesion (GO:0022610)                                  7
Metabolic process (GO:0008152)                                   56
Immune system process (GO:0002376)                                8

(c)

                                                              Cellular
                                                              component
Cell junction (GO:0030054)                                        1
Membrane (GO:0016020)                                             3
Macromolecular complex (GO:0032991)                              12
Cell part (GO:0044464)                                           17
Organelle (GO:0043226)                                           11
Extracellular region (GO:0005576)                                 1

(d)

                                                               Protein
                                                                class

Protease (PC00190)                                                1
Cytoskeletal protein (PC00085)                                    8
Transporter (PC00227)                                             2
Transferase (PC00220)                                             6
Oxidoreductase (PC00176)                                          6
Lyase (PC00144)                                                   1
Cell adhesion molecule (PC00069)                                  3
Ligase (PC00142)                                                  7
Nucleic acid binding (PC00171)                                   23
Signaling molecule (PC00207)                                      7
Enzyme modulator (PC00095)                                        6
Calcium-binding protein (PC00060)                                 2
Defense/immunity protein (PC00090)                                1
Hydrolase (PC00121)                                               5
Transfer/carrier protein (PC00219)                                1
Membrane traffic protein (PC00150)                                5
Phosphatase (PC00181)                                             1
Transcription factor (PC00218)                                    7
Chaperone (PC00072)                                               3
Kinase (PC00137)                                                  2
Storage protein (PC00210)                                         4
Isomerase (PC00135)                                               2
Receptor (PC00197)                                                6

Note: Table made from bar graph.

Figure 6: GO terms distribution in the molecular function (a), the
biological process (b), the cellular components (c), and the protein
class (d) for postnatal ESTs.

(a)

                                                              Molecular
                                                              functions

Transporter activity (GO:0005215)                                 3
Translation regulator activity (GO:0045182)                       3
Enzyme regulator activity (GO:0030234)                            7
Catalytic activity (GO:0003824)                                  25
Receptor activity (GO:0004872)                                    1
Nucleic acid binding transcription factor activity                8
(GO:0001071)
Antioxidant activity (GO:0016209)                                 1
Structural molecule activity (GO:0005198)                        16
Binding (GO:0005488)                                             34

(b)

                                                             Biological
                                                              process

Cellular component organization or biogenesis (GO:0071840)       11
Cellular process (GO:0009987)                                    20
Localization (GO:0051179)                                        11
Apoptotic process (GO:0006915)                                    3
Reproduction (GO:0000003)                                         3
Biological regulation (GO:0065007)                               16
Response to stimulus (GO:0050896)                                 5
Developmental process (GO:0032502)                                8
Multicellular organismal process (GO:0032501)                     3
Biological adhesion (GO:0022610)                                  1
Metabolic process (GO:0008152)                                   55
Immune system process (GO:0002376)                                7

(c)

                                                              Cellular
                                                              component

Membrane (GO:0016020)                                             2
Macromolecular complex (GO:0032991)                               4
Extracellular matrix (GO:0031012)                                 2
Cell part (GO:0044464)                                            9
Organelle (GO:0043226)                                            7
Extracellular region (GO:0005576)                                 2

(d)

                                                               Protein
                                                                class

Extracellular matrix protein (PC00102)                            2
Cytoskeletal protein (PC00085)                                    4
Transporter (PC00227)                                             4
Transferase (PC00220)                                             5
Oxidoreductase (PC00176)                                          5
Lyase (PC00144)                                                   1
Cell adhesion molecule (PC00069)                                  2
Ligase (PC00142)                                                  1
Nucleic acid binding (PC00171)                                   20
Signaling molecule (PC00207)                                      3
Enzyme modulator (PC00095)                                        8
Calcium-binding protein (PC00060)                                 1
Defense/immunity protein (PC00090)                                2
Hydrolase (PC00121)                                               4
Transfer/carrier protein (PC00219)                                2
Membrane traffic protein (PC00150)                                3
Phosphatase (PC00181)                                             1
Transcription factor (PC00218)                                    8
Chaperone (PC00072)                                               2
Structural protein (PC00211)                                      1
Kinase (PC00137)                                                  1
Storage protein (PC00210)                                         2
Isomerase (PC00135)                                               2
Receptor (PC00197)                                                1

Note: Table made from bar graph.
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Article Details
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Title Annotation:Research Article
Author:Ozgenturk, Nehir Ozdemir; Ulu, Zehra Omeroglu; Ulu, Salih; Un, Cemal; Oztabak, Kemal Ozdem; Altunatm
Publication:International Journal of Genomics
Article Type:Report
Geographic Code:7TURK
Date:Jan 1, 2017
Words:5615
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