• Korean Journal of Breeding Science
• /
• v.42 no.4
• /
• pp.365-373
• /
• 2010

TILLING (Targeting Induced Local Lesions IN Genomes) is broadly regarded as an excellent methodology for reverse genetics applications. Approximately 15,000 $M_3$ TILLING lines have been developed via the application of gamma-ray irradiation to rice seeds (cv. Donganbyeo), followed by subsequent selections. In an effort to evaluate the genetic diversity of the TILLING population, we have employed the AFLP multiple dominant marker technique. A total of 96 (0.64%) TILLING lines as well as Donganbyeo were selected randomly and their genetic diversity was assessed based on AFLP marker polymorphisms using 5 primer combinations. An average of 100.4 loci in a range of 97 to 106 was detected using these primer combinations, yielding a total of 158 (31.4%) polymorphic loci between Donganbyeo and each of the 96 lines. A broad range of similarity from 80% to 96% with an average of 89.4% between Donganbyeo and each of the 96 lines was also observed, reflecting the genetic diversity of the TILLING population. Approximately 28 polymorphic loci have been cloned and their sequences were BLAST-searched against rice whole genome sequences, resulting in 20 matches to each of the gene bodies including exon, intron, 1 kb upstream and 1 kb downstream regions. Six polymorphic loci evidenced changes in the coding regions of genes as compared to the rice pseudomolecules, 4 loci of which exhibited missense mutations and 2 loci of which exhibited silent mutations. Therefore, the results of our study show that the TILLING rice population should prove to be a useful genetic material pool for functional genomics as well as mutation breeding applications.

• Korean Journal of Breeding Science
• /
• v.42 no.1
• /
• pp.1-10
• /
• 2010

Five mutants were investigated at the molecular level to determine the factors responsible for mutated endosperm types. They were classified as high (HA) or low amylose (LA) phenotypes based on the amylose content in endosperm. The five were previously produced from Ilpum and Shindongjin cultivar treated with N-methyl-N-nitrosourea and gamma-ray irradiation, respectively. Analysis of the genomic structure and expression of Granule-bounded Starch Synthase I (GBSSI) genes revealed that mutants generally showed a higher incidence of nucleotide transition than transversion, and the $A:T{\rightarrow}G:C$ transition was particularly prevalent. The rates of nucleotide substitution in HA mutants were generally higher than those in the LA mutants, leading to higher substitutions of amino acid in the HA mutants. Neither nucleotide substitutions interfering with intron splicing or causing early termination of protein translation were found, nor any large-sized deletions or additions were found in all the mutants. In principle, amylose content can be regulated by three factors: internal alterations of GBSSI protein, the strength of gene expression, and other unknown external factors. Our results indicate that the endosperm mutants from Shindongjin arose from internal alterations of GBSSI proteins, which may be the result of amino acid substitutions. On the other hand, the Ilpum mutants might be principally caused by the alteration of gene expression level. Analysis of another three glutinous cultivars revealed that the major factor leading to glutinous phenotypes is the 23-bp duplicative motif (5'-ACGGGTTCCAGGGCCTCAAGCCC-3') commonly found in exon 2, which results in the premature termination of protein translation leading to the production of a non-functional GBSSI enzyme.

• Annals of Pediatric Endocrinology and Metabolism
• /
• v.23 no.4
• /
• pp.235-239
• /
• 2018

Most cases of congenital hyperthyroidism are autoimmune forms caused by maternal thyroid stimulating antibodies. Nonautoimmune forms of congenital hyperthyroidism caused by activating mutations of the thyrotropin receptor (TSHR) gene are rare. A woman gave birth to a boy during an emergency cesarean section at 33 weeks of gestation due to fetal tachycardia. On the 24th day of life, thyroid function tests were performed due to persistent tachycardia, and hyperthyroidism was confirmed. Auto-antibodies to TSHR, thyroid peroxidase, and thyroglobulin were not found. The patient was treated with propylthiouracil and propranolol, but hyperthyroidism was not well controlled. At 3 months of age, the patient had craniosynostosis and hydrocephalus, and underwent a ventriculoperitoneal shunt operation. Direct sequencing of the TSHR gene showed a heterozygous mutation of c.1899C>A (p.Asp633Glu) in exon 10. No mutations were discovered in any of the parents in a familial genetic study. We have reported a case of sporadic nonautoimmune congenital hyperthyroidism, by a missense mutation of the TSHR gene, for the first time in South Korea.

• Korean Journal of Poultry Science
• /
• v.45 no.4
• /
• pp.291-298
• /
• 2018

TBC1D1 gene has known functional effects on body energy homeostasis and glucose uptake pathway in skeletal muscle tissue. This biological function is reported to have significant effects on traits of growth and meat quality in chicken. In this study, we focused on two single nucleotide polymorphisms (SNPs) (g.70179137A>G and g.70175861T>C) identified through SNP annotation information of Korean native chicken and previous literature for TBC1D1 in chicken. Association of SNPs in TBC1D1 with growth and serum clinical-chemical traits were evaluated. A total of 584 male and female birds from five Korean native chicken lines were used in the study. The SNP1 (g.70179137A>G) is located in intron 11 and SNP2 (g.70175861T>C) is a non-synonymous missense mutation in exon 10, responsible for the amino acid change from Methionine to Valine. The A allele of SNP1 and T allele of SNP2 had the highest allele frequencies. Both SNPs indicated moderate polymorphism information content values (0.25

• Asian-Australasian Journal of Animal Sciences
• /
• v.32 no.6
• /
• pp.891-895
• /
• 2019

Objective: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the ${\alpha}$-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). Methods: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). Results: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources ($0.25ng/{\mu}L$). Conclusion: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

• Laboraroty Animal Research
• /
• v.34 no.4
• /
• pp.279-287
• /
• 2018

Placenta specific 8 (PLAC8, also known as ONZIN) is a multi-functional protein that is highly expressed in the intestine, lung, spleen, and innate immune cells, and is involved in various diseases, including cancers, obesity, and innate immune deficiency. Here, we generated a Plac8 knockout mouse using the CRISPR/Cas9 system. The Cas9 mRNA and two single guide RNAs targeting a region near the translation start codon at Plac8 exon 2 were microinjected into mouse zygotes. This successfully eliminated the conventional translation start site, as confirmed by Sanger sequencing and PCR genotyping analysis. Unlike the previous Plac8 deficient models displaying increased adipose tissue and body weights, our male Plac8 knockout mice showed rather lower body weight than sex-matched littermate controls, though the only difference between these two mouse models is genetic context. Differently from the previously constructed embryonic stem cell-derived Plac8 knockout mouse that contains a neomycin resistance cassette, this knockout mouse model is free from a negative selection marker or other external insertions, which will be useful in future studies aimed at elucidating the multi-functional and physiological roles of PLAC8 in various diseases, without interference from exogenous foreign DNA.

• Journal of Medicine and Life Science
• /
• v.17 no.3
• /
• pp.65-73
• /
• 2020

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a single-gene disease of the cerebral small blood vessels caused by mutations in the NOTCH3 gene on chromosome 19. Although CADASIL was known as a rare disease, recent research has suggested that the NOTCH variants could be found frequently even in the general population. The main clinical features included recurrent stroke, migraine, psychiatric symptoms, and progressive cognitive decline. On brain magnetic resonance imaging, patients with CADASIL showed multifocal white matter hyperintensity lesions, lacunar infarcts, microbleeds, and brain atrophy. Among them, lacunar infarcts and brain atrophy are important in predicting the clinical outcomes of patients with CADASIL. In the Jeju National University Hospital, we have diagnosed 213 CADASIL patients from 2004 to 2020. Most NOTCH3 mutations were located in exon 11 (94.4%), and p.Arg544Cys was the most common mutation. The mean age at diagnosis was 61.0±12.8 years. The most common presenting symptoms were ischemic stroke (24.4%), followed by cognitive impairment(15.0%), headache (8.9%), and dizziness(8.0%). Although the exact prevalence of CADASIL in Jeju is still unknown, the disease prevalence could be as high as 1% of the population considering the prevalence reported in Taiwan. Therefore, it is necessary to discover efficient biomarkers and genetic tests that can accurately screen and diagnose patients suspected of having CADASIL in this region. Ultimately, it is urgent to explore the exact pathogenesis of the disease to identify leading substances of treatment potential, and for this, multi-disciplinary research through active support from the Jeju provincial government as well as the national government is essential.

• Journal of Animal Science and Technology
• /
• v.63 no.2
• /
• pp.248-261
• /
• 2021

Attributable to their major function in pathogen recognition, the use of bovine leukocyte antigens (BoLA) as disease markers in immunological traits in cattle is well established. However, limited report exists on polymorphism of the BoLA gene in zebu cattle breeds by high resolution typing methods. Thus, we used a polymerase chain reaction sequence-based typing (PCR-SBT) method to sequence exon 2 of the BoLA class II DRB3 gene from 100 animals (Boran, n = 13; Sheko, n = 20; Fogera, n = 16; Horro, n = 19), Hanwoo cattle (n = 18) and Bangladesh Red Chittagong zebu (n = 14). Out of the 59 detected alleles, 43 were already deposited under the Immuno Polymorphism Database for major histocompatibility complex (IPD-MHC) while 16 were unique to this study. Assessment of the level of genetic variability at the population and sequence levels with genetic distance in the breeds considered in this study showed that Zebu breeds had a gene diversity score greater than 0.752, nucleotide diversity score greater than 0.152, and mean number of pairwise differences higher than 14, being very comparable to those investigated for other cattle breeds. Regarding neutrality tests analyzed, we investigated that all the breeds except Hanwoo had an excess number of alleles and could be expected from a recent population expansion or genetic hitchhiking. Howbeit, the observed heterozygosity was not significantly (p < 0.05) higher than the expected heterozygosity. The Hardy Weinberg equilibrium (HWE) analysis revealed non-significant excess of heterozygote animals, indicative of plausible over-dominant selection. The pairwise FST values suggested a low genetic variation among all the breeds (FST = 0.056; p < 0.05), besides the rooting from the evolutionary or domestication history of the cattle. No detached clade was observed in the evolutionary divergence study of the BoLA-DRB3 gene, inferred from the phylogenetic tree based on the maximum likelihood model. The investigation herein indicated the clear differences in BoLA-DRB3 gene variability between African and Asian cattle breeds.

• Korean Journal of Veterinary Service
• /
• v.44 no.4
• /
• pp.185-194
• /
• 2021

Polyglutamine extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyglutamine aggregates in Huntington's disease (HD). Mutant huntingtin can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. To better understand the mechanism by which an elongated polyglutamine causes aggregates, we have developed an in vitro binding assay system of polyglutamine tract from truncated huntingtin. We made GST-HD exon1 fusion proteins which have expanded polyglutamine epitopes (e.g., 17, 23, 32, 46, 60, 78, 81, and 94 CAG repeats). In the present emergence of new study adjusted nanotechnology on protein chip such as surface plasmon resonance strategy which used to determine the substance which protein binds in drug discovery platform is worth to understand better neurodegenerative diseases (i.e., Alzheimer disease, Parkinson disease and Huntington disease) and its pathogenesis along with development of therapeutic measures. Hence, we used strengths of surface plasmon resonance (SPR) technology which is enabled to examine binding specificity and explore targeted molecular epitope using its electron charged wave pattern in HD pathogenesis utilize conjugated mutant epitope of HD protein and its interaction whether wild type GST-HD interacts with mutant GST-HD with maximum binding affinity at pH 6.85. We found that the maximum binding affinity of GST-HD17 with GST-HD81 was higher than the binding affinities of GST-HD17 with other mutant GST-HD constructs. Furthermore, our finding illustrated that the mutant form of GST-HD60 showed a stronger binding to GST-HD23 or GST-HD17 than GST-HD60 or GST-HD81. These results indicate that the binding affinity of mutant huntingtin does not correlate with the length of polyglutamine. It suggests that the aggregation of an expanded polyglutamine might have easily occurred in the presence of wild type form of huntingtin.

• Journal of Ginseng Research
• /
• v.46 no.4
• /
• pp.505-514
• /
• 2022

Background: The roots of Panax ginseng contain two types of tetracyclic triterpenoid saponins, namely, protopanaxadiol (PPD)-type saponins and protopanaxatiol (PPT)-type saponins. In P. ginseng, the protopanaxadiol 6-hydroxylase (PPT synthase) enzyme catalyses protopanaxatriol (PPT) production from protopanaxadiol (PPD). In this study, we constructed homozygous mutant lines of ginseng by CRISPR/Cas9-mediated mutagenesis of the PPT synthase gene and obtained the mutant ginseng root lines having complete depletion of the PPT-type ginsenosides. Methods: Two sgRNAs (single guide RNAs) were designed for target mutations in the exon sequences of the two PPT synthase genes (both PPTa and PPTg sequences) with the CRISPR/Cas9 system. Transgenic ginseng roots were generated through Agrobacterium-mediated transformation. The mutant lines were screened by ginsenoside analysis and DNA sequencing. Result: Ginsenoside analysis revealed the complete depletion of PPT-type ginsenosides in three putative mutant lines (Cr4, Cr7, and Cr14). The reduction of PPT-type ginsenosides in mutant lines led to increased accumulation of PPD-type ginsenosides. The gene editing in the selected mutant lines was confirmed by targeted deep sequencing. Conclusion: We have established the genome editing protocol by CRISPR/Cas9 system in P. ginseng and demonstrated the mutated roots producing only PPD-type ginsenosides by depleting PPT-type ginsenosides. Because the pharmacological activity of PPD-group ginsenosides is significantly different from that of PPT-group ginsenosides, the new type of ginseng mutant producing only PPD-group ginsenosides may have new pharmacological characteristics compared to wild-type ginseng. This is the first report to generate target-induced mutations for the modification of saponin biosynthesis in Panax species using CRISPR-Cas9 system.