Discovery of cSNPs in Pig Using Full-length Enriched cDNA Libraries of the Korean Native Pig as a Source of Genetic Diversity

Dirisala, Vijaya R.,Kim, Ju-Hyun,Park, Kwang-Ha,Lee, Hoon-Taek,Park, Chan-Kyu Korean Society for Biotechnology and Bioengineerin 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

Clones from full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We analyzed 3,210 chromatograms obtained from sequencing the 5'-ends of brainstem, liver, neocortex, and spleen clones derived from full-length enriched cDNA libraries of Korean native pigs. In addition, 50,000 pig EST sequence trace files were obtained from Genbank and combined with our sequencing information for SNP identification in silica. For the SNP analysis, neocortex, and liver libraries were newly constructed, whereas the sequencing results from brainstem and spleen libraries were from previously constructed libraries. The putative SNPs from the in silica analysis were confirmed by genomic PCR from a group of 20 pigs of four different breeds. Using this approach, 86% of cSNPs identified in silico were confirmed and the SNP detection frequency was 1 SNP per 338 bp. Interestingly, we found a valine deletion at amino acid position 126 of the neuronal and endocrine protein gene in the Korean native pig. We confirmed that this deletion was caused by alternative splicing at the NAGNAG acceptors. Our study shows that large-scale EST sequencing of Korean native pigs can be effectively employed for natural polymorphism-based pig genome analysis.

Reporting 678 putative cSNPs from full-length enriched cDNA sequences of the Korean native pig

Park, K.,Dirisala, V.R.,Oh, Y.,Choi, H.,Lee, K.T.,Kim, J.H.,Lee, H.T.,Seo, K.H.,Park, C. Blackwell Publishing Ltd 2009 Journal of animal breeding and genetics Vol.126 No.2

<P>Summary</P><P>Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We have analysed 1970 high-quality chromatograms (Phred value ≥ 30) that were obtained from sequencing the 5′ ends of brainstem, liver, neocortex and spleen clones derived from full-length enriched cDNA libraries from Korean native pigs. In addition, 50 000 pig expressed sequence tag (EST) sequence trace files were obtained from Genbank and combined with our sequencing information to facilitate SNP identification <I>in silico</I>. The process generated 8118 contigs, of which 239 included minimum one sequence from Korean native pig and contained 678 putative coding single nucleotide polymorphisms (cSNPs). Of these, 33 putative cSNPs were randomly selected for confirmatory analysis and validated using 20 pigs from four different breeds (Duroc, Landrace, Yorkshire, Korean native pig). Of the 33 putative cSNPs, 20 were confirmed (61%), which was similar to the frequency reported in other studies. We also identified 15 new cSNPs from the validation process, which were not detected by our <I>in silico</I> analysis. Our study shows that analysing genetically diverse pig breeds including the Korean native pig could serve as a useful strategy for generating a large number of cSNPs.</P>

Discovery of cSNPs in Pig UsingFull-length Enriched cDNA Libraries of theKorean Native Pig as a Source of GeneticDiversity

박찬규,이훈택,Vijaya R. Dirisala,Juhyun Kim,Kwangha Park 한국생물공학회 2007 Biotechnology and Bioprocess Engineering Vol.12 No.4

Clones from full-length enriched cDNA libraries serve as valuable resources for functional genomic studies. We analyzed 3,210 chromatograms obtained from sequencing the 5'-ends of brainstem, liver, neocortex, and spleen clones derived from full-length enriched cDNA libraries of Korean native pigs. In addition, 50,000 pig EST sequence trace files were obtained from Genbank and combined with our sequencing information for SNP identification in silico. For the SNP analysis, neocortex, and liver libraries were newly constructed, whereas the sequencing results from brainstem and spleen libraries were from previously constructed libraries. The putative SNPs from the in silico analysis were confirmed by genomic PCR from a group of 20 pigs of four different breeds. Using this approach, 86% of cSNPs identified in silico were confirmed and the SNP detection frequency was 1 SNP per 338 bp. Interestingly, we found a valine deletion at amino acid position 126 of the neuronal and endocrine protein gene in the Korean native pig. We confirmed that this deletion was caused by alternative splicing at the NAGNAG acceptors. Our study shows that large-scale EST sequencing of Korean native pigs can be effectively employed for natural polymorphism-based pig genome analysis.

Identification of 1,531 cSNPs from Full-length Enriched cDNA Libraries of the Korean Native Pig Using in Silico Analysis

오윤신,Dinh Truong Nguyen,박광하,Vijaya R. Dirisala,최호준,박찬규 한국유전체학회 2009 Genomics & informatics Vol.7 No.2

Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomics related studies, genome annotation and SNP discovery. We analyzed 7,392 high-quality chromatograms (Phred value ≥30) obtained from sequencing the 5' ends of clones derived from full-length enriched cDNA libraries of Korean native pigs including brainstem, liver, cerebellum, neocortex and spleen libraries. In addition, 50,000 EST sequence trace files obtained from GenBank were combined with our sequences to identify cSNPs in silico. The process generated 11,324 contigs, of which 2,895 contigs contained at least one SNP and among them 610 contigs had a minimum of one sequence from Korean native pigs. Of 610 contigs, we randomly selected 262 contigs and performed in silico analysis for the identification of cSNPs. From the results, we identified 1,531 putative coding single nucleotide polymorphisms (cSNPs) and the SNP detection frequency was one SNP per 465 bp. A large-scale sequencing result of clones from full-length enriched cDNA libraries and identified cSNPs will serve as a useful resource to functional genomics related projects such as a pig HapMap project in the near future.

Identification of Genes Differentially Expressed in Wild Type and Purkinje Cell Degeneration Mice

박찬규,Rui Xiao,Youngsook Park,Vijaya R. Dirisala,Ya-Ping Zhang,엄상준,이훈택 한국분자세포생물학회 2005 Molecules and cells Vol.20 No.2

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin-28k, paravalbumin, matrix gamma-carboxyglutamate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.

A Simple, Rapid, Efficient and Inexpensive Strategy for Sequencing Clones from cDNA Libraries

Nguyen, Dinh Truong,Oh, Youn-Shin,Dirisala, Vijaya R.,Choi, Ho-Jun,Park, Keun-Kyu,Kim, Jin-Hoi,Park, Chan-Kyu 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.5

In this report, we describe a simple, rapid, efficient and inexpensive strategy for sequencing inserted DNAs from clones of cDNA or gDNA libraries. This strategy uses PCR products directly amplified from transformed bacterial colonies, with universal primers within the vector. The method can be applied for sequencing cDNA or gDNA libraries with up to 4 ~ 5 kb insert sizes, without overnight liquid culture or plasmid DNA preparation steps. We successfully used this method to analyze clones from full-length, enriched cDNA libraries. Although simple, following this strategy will significantly help researchers to avoid unnecessary steps in the analysis of a cDNA library.

Molecular characterization of the human ABO blood group orthologus system in pigs

Nguyen, D. T.,Choi, H.,Jo, H.,Kim, J.‐,H.,Dirisala, V. R.,Lee, K.‐,T.,Kim, T.‐,H.,Park, K.‐,K.,Seo, K.,Park, C. Blackwell Publishing Ltd 2011 Animal genetics Vol.42 No.3

<P><B>Summary</B></P><P>The selection and use of animals with blood group 0 in the process of transplanting pig organs or tissues into humans can positively contribute to the control of acute immune rejection due to differences in blood groups. Exon‐specific PCRs for the <I>porcine blood group A transferase</I> gene against genomic DNA from either blood group A or 0 animals resulted in the amplification failure of the <I>A0 blood group</I> gene exon 8 from blood group 0 animals. To characterize the genetic abnormality in the genome of blood group 0 animals, we screened bacterial artificial chromosome (BAC) clones from a Korean native pig BAC library which had the blood group 0 allele, and carried out shotgun sequencing. The analysis showed that the 0 allele has a large deletion between exon 7 of the <I>A0</I> blood group gene and the neighbouring <I>SURF6</I>. We also showed that the ABO blood group antigens in humans and the A0 blood group antigens in pigs are coded by mutations within the orthologous <I>glycosyltransferase</I> gene. In addition, we developed a multiplex genotyping method for the porcine <I>A0</I> blood group gene.</P>

Identification of 1,531 cSNPs from Full-length Enriched cDNA Libraries of the Korean Native Pig Using in Silico Analysis

Oh, Youn-Shin,Nguyen, Dinh Truong,Park, Kwang-Ha,Dirisala, Vijaya R.,Choi, Ho-Jun,Park, Chan-Kyu Korea Genome Organization 2009 Genomics & informatics Vol.7 No.2

Sequences from the clones of full-length enriched cDNA libraries serve as valuable resources for functional genomics related studies, genome annotation and SNP discovery. We analyzed 7,392 high-quality chromatograms (Phred value ${\geq}$30) obtained from sequencing the 5' ends of clones derived from full-length enriched cDNA libraries of Korean native pigs including brainstem, liver, cerebellum, neocortex and spleen libraries. In addition, 50,000 EST sequence trace files obtained from GenBank were combined with our sequences to identify cSNPs in silico. The process generated 11,324 contigs, of which 2,895 contigs contained at least one SNP and among them 610 contigs had a minimum of one sequence from Korean native pigs. Of 610 contigs, we randomly selected 262 contigs and performed in silico analysis for the identification of cSNPs. From the results, we identified 1,531 putative coding single nucleotide polymorphisms (cSNPs) and the SNP detection frequency was one SNP per 465 bp. A large-scale sequencing result of clones from full-length enriched cDNA libraries and identified cSNPs will serve as a useful resource to functional genomics related projects such as a pig HapMap project in the near future.

Molecular insights into the role of genetic determinants of congenital hypothyroidism

Kollati, Yedukondalu,Akella, Radha Rama Devi,Naushad, Shaik Mohammad,Patel, Rajesh K.,Reddy, G. Bhanuprakash,Dirisala, Vijaya R. Korea Genome Organization 2021 Genomics & informatics Vol.19 No.3

In our previous studies, we have demonstrated the association of certain variants of the thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase (TPO), and thyroglobulin (TG) genes with congenital hypothyroidism. Herein, we explored the mechanistic basis for this association using different in silico tools. The mRNA 3'-untranslated region (3'-UTR) plays key roles in gene expression at the post-transcriptional level. In TSHR variants (rs2268477, rs7144481, and rs17630128), the binding affinity of microRNAs (miRs) (hsa-miR-154-5p, hsa-miR-376a-2-5p, hsa-miR-3935, hsa-miR-4280, and hsa-miR-6858-3p) to the 3'-UTR is disrupted, affecting post-transcriptional gene regulation. TPO and TG are the two key proteins necessary for the biosynthesis of thyroid hormones in the presence of iodide and H₂O₂. Reduced stability of these proteins leads to aberrant biosynthesis of thyroid hormones. Compared to the wild-type TPO protein, the p.S398T variant was found to exhibit less stability and significant rearrangements of intra-atomic bonds affecting the stoichiometry and substrate binding (binding energies, ΔG of wild-type vs. mutant: -15 vs. -13.8 kcal/mol; and dissociation constant, K_d of wild-type vs. mutant: 7.2E^-12 vs. 7.0E^-11 M). The missense mutations p.G653D and p.R1999W on the TG protein showed altered ΔG(0.24 kcal/mol and 0.79 kcal/mol, respectively). In conclusion, an in silico analysis of TSHR genetic variants in the 3'-UTR showed that they alter the binding affinities of different miRs. The TPO protein structure and mutant protein complex (p.S398T) are less stable, with potentially deleterious effects. A structural and energy analysis showed that TG mutations (p.G653D and p.R1999W) reduce the stability of the TG protein and affect its structure-functional relationship.

Extraction and characterization of collagen from the skin of Pterygoplichthys pardalis and its potential application in food industries

Ramesh Nurubhasha,N. S. Sampath Kumar,Satish K. Thirumalasetti,G. Simhachalam,Vijaya R. Dirisala 한국식품과학회 2019 Food Science and Biotechnology Vol.28 No.6

The primary objective of this study was toextract collagen from underutilized fish species owing to itscost effective nature and also its ability to address thedemand of type I collagen arising from food and pharmaceuticalindustries. Acid and pepsin soluble collagen(ppASC and ppPSC) were extracted from the skin of suckercatfish (Pterygoplichthys pardalis) with a yield of 19.6 and23.8% on wet weight basis respectively. The same werecharacterized and confirmed as type I collagen by SDS–PAGE, FTIR and UV–Vis spectroscopy, amino acid analysis,and Zeta potential. Taking into consideration theapplication of collagen in food industry, a food product wasdeveloped by incorporating with fresh cheese. This fortificationwas found to be acceptable and had not altered thetaste, odor and other sensory properties of the product.