RESUMEN
DIO3 gene encoding type 3 iodothyronine deiodinase is an imprinted gene, located in the DLK1-DIO3 (delta-like 1 homolog-type 3 iodothyronine deiodinase) imprinted domain, and is potentially involved in degrading excessive amounts of thyroid hormone to protect embryogenesis. However, the underlying regulatory mechanism of the imprinted DIO3 gene expression during fetal and neonatal development in goats has not been elucidated. In this study, we explored the DNA methylation patterns of the caprine DIO3 intragenic CpG island and quantified gene expression level in six tissues from Chinese Nanjiang Yellow 3-day old kids. The expression of the DIO3 gene was determined using quantitative reverse transcription-polymerase chain reactions (qRT-PCRs), while the identification of methylation patterns was determined using bisulfite-sequencing PCRs. Modest, and non-significant (P > 0.05), methylation patterns were noted for the DIO3 CpG island methylation in the brain, heart, liver, kidney, lung, and longissimus dorsi tissues (ranging from 26.48 to 34.92%). The expression level of the DIO3 mRNA was significantly higher (P < 0.05) in the liver tissue than in the other five tissues. Pearson's correlation analysis revealed that there was no significant relationship between methylation and gene expression (P > 0.05), which indicated that the expression of the caprine DIO3 gene was likely modified by other regulatory elements. This study identified DNA methylation and expression patterns of the DIO3 gene in goats and provided insights into further regulatory mechanisms of expression and imprinting in the DLK1-DIO3 domain.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Cabras/genética , Yoduro Peroxidasa/genética , Hígado/enzimología , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Islas de CpG , Cabras/crecimiento & desarrollo , Cabras/metabolismo , Corazón/crecimiento & desarrollo , Yoduro Peroxidasa/metabolismo , Riñón/enzimología , Riñón/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Pulmón/enzimología , Pulmón/crecimiento & desarrollo , Músculo Esquelético/enzimología , Músculo Esquelético/crecimiento & desarrolloRESUMEN
Interleukin 18 (IL-18), as a member of IL-1 superfamily, is an important pleiotropic cytokine that modulates Th1 immune responses. In this report, we cloned and identified a homolog of IL-18 in giant panda (Ailuropoda melanoleuca) (designated as AmIL-18) from peripheral blood mononuclear cells stimulated with lipopolysaccharide. The open readin g frame of AmIL-18 cDNA is 579 bp encoding a deduced protein of 192 amino acids. AmIL-18 gDNA fragments contained 5 exons and 4 introns. The amino acid sequence of AmIL-18 shared 23.9 to 87.0% identity with other species. To evaluate the effects of AmIL-18 on the immune response, we expressed the recombinant AmIL-18 in Escherichia coli BL21 (DE3). The fusion protein PET-AmIL-18 was purified by nickel affinity column chromatography and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The biological function of purified PET-AmIL-18 was determined on mouse splenocytes by quantitative real-time polymerase chain reaction. INF-γ and other cytokines were increased when stimulated by PET-AmIL-18, particularly when combined with recombinant human interleukin 12, while a Th2-type cytokine, interleukin-4, was strikingly suppressed. These results will provide information for the potential use of recombinant proteins to manipulate the immune response in giant pandas and facilitate the study to protect this treasured species.
Asunto(s)
Interleucina-18/genética , Leucocitos Mononucleares/inmunología , Sistemas de Lectura Abierta , Ursidae/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Femenino , Expresión Génica , Humanos , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Interleucina-12/farmacología , Interleucina-18/inmunología , Interleucina-4/biosíntesis , Interleucina-4/metabolismo , Intrones , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Lipopolisacáridos/farmacología , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Ursidae/inmunologíaRESUMEN
Y-chromosome short tandem repeats (Y-STRs) are useful tools for identifying paternity origin and male-female mixed samples because of their male-specificity, haploid inheritance and relatively simplicity. We focused on novel Y-STRs deposited in the human Genome database from DYS708 to DYS726. We typed 16 male-specific Y-STRs from males of a Chinese Han population residing in Shanxi Province (north China), including DYS708-719, DYS721-723, and DYS726, but failed in typing DYS720, DYS724 and DYS725. The 16 Y-STRs, with mean gene diversity (GD) of 0.79, included three trinucleotide Y-STRs (711, 718, 719), nine tetranucleotide STRs (708, 709, 710, 712, 713, 715, 722, 723, 726) and four pentanucleotide repeat STRs (714, 716, 717, 721). DYS712, consisting of eight alleles, was the most informative STR in our population, with a GD of 0.843. The STRs were classified as simple STRs and complex STRs, according to their structures based on sequencing. Genetic indexes, including allele frequencies, haplotype distribution and male-specificity were determined. The Y-STRs, especially those male-specific, tetra- and penta-nucleotide, with only one copy on Y-chromosome, and relative simple structures, such as DYS709, DYS714, DYS715, DYS716, DYS718, DYS719, and DYS726, were suggested for the future forensic DNA analysis, while DYS724 and DYS725 were not recommended for their multi-copy distribution. The population data provided putative Y-STRs for future genetic and forensic applications.