RESUMO
Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.
Assuntos
Fucosiltransferases/genética , Glicoesfingolipídeos/biossíntese , Regiões Promotoras Genéticas , Suínos/genética , Animais , Ilhas de CpG , Metilação de DNA , Fucosiltransferases/metabolismo , Ativação Transcricional , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Biologists and scientists can use the data from Alzheimer's disease (AD) gene expression microarrays to mine AD disease-related genes. Because of disadvantages such as small sample sizes, high dimensionality, and a high level of noise, it is difficult to obtain accurate and meaningful biological information from gene expression profiles. In this paper, we present a novel approach for utilizing AD microarray data to identify the morbigenous genes. The Fisher score, a classical feature selection method, is utilized to evaluate the importance of each gene. Genes with a large between-classes variance and small within-class variance are selected as candidate morbigenous genes. The results using an AD dataset show that the proposed approach is effective for gene selection. Satisfactory accuracy can be achieved by using only a small number of selected genes.
Assuntos
Algoritmos , Doença de Alzheimer/genética , Modelos Genéticos , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , HumanosRESUMO
The bactericidal/permeability-increasing protein (BPI) gene has been identified as a candidate gene for disease-resistance breeding. We evaluated whether polymorphisms in exons 4 and 10 of the BPI gene are associated with immune indices [interleukin-2 (IL-2), IL-4, IL-6, interferon-b (IFN-b), IL-10, and IL-12]. In this study, we identified one mutation (C522T) in the BPI exon 4 site and two mutations (A1060G and T1151G) in the BPI exon 10 site. Correlation analysis revealed that in the Sutai pig population, the effect of genotypes at the BPI exon 4 site on the level of IL-6 was significant (P < 0.05), with an effective genotype of CD; moreover, the effect of genotypes at the BPI exon 10 site on the level of IL-12 was significant (P < 0.05), and the effective genotype was AB. The optimal combined genotype was CD-AB, which was more effective regarding the IL-6 and IL-12 levels compared to the other combined genotypes (P < 0.05). These results indicate that single nucleotide polymorphisms and the combined genotypes of BPI exons 4 and 10 affect immune indices in Sutai pigs. Therefore, these genotypes should be further examined as effective markers for disease-resistant breeding of pigs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Polimorfismo de Nucleotídeo Único , Suínos/imunologia , Animais , Resistência à Doença , Éxons , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , Seleção Artificial , Suínos/genéticaRESUMO
The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Proteínas Sanguíneas/biossíntese , Resistência à Doença/genética , Infecções por Escherichia coli/genética , Escherichia coli/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Suscetibilidade a Doenças/microbiologia , Suscetibilidade a Doenças/veterinária , Endotoxinas/genética , Endotoxinas/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/veterinária , Vetores Genéticos , Genótipo , Suínos , DesmameRESUMO
TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.
Assuntos
Resistência à Doença/genética , Infecções por Escherichia coli/genética , Suínos/genética , Receptor 4 Toll-Like/genética , Transcrição Gênica , Animais , Animais Endogâmicos , Regulação para Baixo , Infecções por Escherichia coli/imunologia , Estudos de Associação Genética , Imunidade Inata/genética , Especificidade de Órgãos , População/genética , Receptor 4 Toll-Like/metabolismoRESUMO
We compared and analyzed the expression of the BPI gene of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 by the real-time PCR method, in order to determine if it is involved in protection against disease caused by ETEC F18. There was a significant difference between 18 and 35-day expression in the jejunum. There were also significant differences between 35-day expression and expression at the other development stages in the duodenum. There were no significant differences in expression at 8, 18, and 30 days in the jejunum. We conclude that the porcine BPI gene may be the direct factor that resisted the ETEC F18 in weaning piglets, and that the resistance to ETEC F18 in weaning piglets is related to up-regulation of mRNA expression of BPI gene to a certain extent.
Assuntos
Envelhecimento/genética , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Regulação da Expressão Gênica no Desenvolvimento , Sus scrofa/crescimento & desenvolvimento , Sus scrofa/genética , Animais , Animais Recém-Nascidos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/metabolismo , Duodeno/metabolismo , Fluorescência , Perfilação da Expressão Gênica , Jejuno/metabolismo , Desnaturação de Ácido Nucleico , Especificidade de Órgãos/genética , Reação em Cadeia da Polimerase , RNA/genética , RNA/metabolismoRESUMO
Complementary DNA (cDNA) is valuable for investigating protein structure and function in the study of life science, but it is difficult to obtain by traditional reverse transcription. We employed a novel strategy to clone human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA, which was directly isolated from the mucous membrane of mouth. The hLIF sequence, which is 609 bp long and is composed of three exons, can be acquired within a few hours by amplifying each exon and splicing all of them using overlap-PCR. This new approach developed is simple, time- and cost-effective, without RNA preparation or cDNA synthesis, and is not limited to the specific tissues for a particular gene and the expression level of the gene.