Genetic variations of mitochondrial antiviral signaling gene (MAVS) in domestic chickens

Mitochondrial antiviral signaling (MAVS) gene plays a key role in antiviral regulation in mammals potentially by activating IRF3/7 and NF-κB and leading to the induction of type I interferon (IFN)-mediated antiviral and inflammatory responses. In this study, we screened genetic polymorphisms of the MAVS gene in various Chinese domestic chicken breeds/populations and evaluated its potential effect on gene expression. Among the sequenced fragment (4678 bp), a total of 75 single nucleotide polymorphisms (SNPs) were identified in 46 chickens from 10 breeds/populations, including 30 coding SNPs and 45 non-coding SNPs. Extremely high haplotype diversity (37 nucleotide haplotypes, 18 amino acid haplotypes) was observed in the coding region (CDS), and a similar pattern of high polymorphisms was also observed for the 3′-untranslated region (3′-UTR). Luciferase assays of two representative 3′-UTR haplotypes were performed in both HEK293 cells and DF-1 chicken fibroblast cells, and we found that they were differentially associated with different abilities on regulating mRNA expression level (P < 0.05). Collectively, we observed a considerably high genetic variability of the MAVS gene, and the 3′-UTR variants had an ability to regulate mRNA expression. These results would cast some clues on understanding the potential role of MAVS on viral resistance in chicken.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

Single nucleotide polymorphisms of myostatin gene in Chinese domestic horses

The myostatin gene (MSTN) is a genetic determinant of skeletal muscle growth. Single nucleotide polymorphisms (SNP) in MSTN are of importance due to their strong associations with horse racing performances. In this study, we screened the SNPs in MSTN gene in 514 horses from 15 Chinese horse breeds. Six SNPs (g.26 T > C, g.156 T > C, g.587A > G, g.598C > T, g.1485C > T, g.2115A > G) in MSTN gene were detected by sequencing and genotyped using PCR-RFLP method. The g.587A > G and g.598C > T residing in the 5′UTR region were novel SNPs identified by this study. The g.2115A > G which have previously been associated with racing performances were present in Chinese horse breeds, providing valuable genetic information for evaluating the potential racing performances in Chinese domestic breeds. The six SNPs together defined thirteen haplotypes, demonstrating abundant haplotype diversities in Chinese horses. Most of the haplotypes were shared among different breeds with no haplotype restricted to a specific region or a single horse breed. AMOVA analysis indicated that most of the genetic variance was attributable to differences among individuals without any significant contribution by the four geographical groups. This study will provide fundamental and instrumental genetic information for evaluating the potential racing performances of Chinese horse breeds.     

Ewing's sarcoma: Analysis of single nucleotide polymorphism in the EWS gene

We aimed to investigate single nucleotide polymorphisms (SNPs) in the EWS gene breaking region in order to analyze Ewing's sarcoma susceptibility. The SNPs were investigated in a healthy subject population and in Ewing's sarcoma patients from Southern Brazil. Genotyping was performed by TaqMan® assay for allelic discrimination using Real-Time PCR. The analysis of incidence of SNPs or different SNP-arrangements revealed a higher presence of homozygote TT-rs4820804 in Ewing's sarcoma patients (p = 0.02; Chi Square Test). About 300 bp from the rs4820804 SNP lies a palindromic hexamer (5′-GCTAGC-3′) and three nucleotides (GTC), which were previously identified to be in close vicinity of the breakpoint junction in both EWS and FLI1 genes. This DNA segment surrounding the rs4820804 SNP is likely to indicate a breakpoint region. If the T-rs4820804 allele predisposes a DNA fragment to breakage, homozygotes (TT-rs4820804) would have double the chance of having a chromosome break, increasing the chances for a translocation to occur. In conclusion, the TT-rs4820804 EWS genotype can be associated with Ewing's sarcoma and the SNP rs4820804 can be a candidate marker to understand Ewing's sarcoma susceptibility.     

Tissue inhibitor of metalloproteinase gene from pearl oyster Pinctada martensii participates in nacre formation

Tissue inhibitors of metalloproteinases (TIMPs) are nature inhibitors of matrix metalloproteinases and play a vital role in the regulation of extracellular matrix turnover, tissue remodeling and bone formation. In this study, the molecular characterization of TIMP and its potential function in nacre formation was described in pearl oyster Pinctada martensii. The cDNA of TIMP gene in P. martensii (Pm-TIMP) was 901 bp long, containing a 5′ untranslated region (UTR) of 51 bp, a 3′ UTR of 169 bp, and an open reading fragment (ORF) of 681 bp encoding 226 amino acids with an estimated molecular mass of 23.37 kDa and a theoretical isoelectric point of 5.42; The predicted amino acid sequence had a signal peptide, 13 cysteine residues, a N-terminal domain and a C-terminal domain, similar to that from other species. Amino acid multiple alignment showed Pm-TIMP had the highest (41%) identity to that from Crassostrea gigas. Tissue expression analysis indicated Pm-TIMP was highly expressed in nacre formation related-tissues, including mantle and pearl sac. After decreasing Pm-TIMP gene expression by RNA interference (RNAi) technology in the mantle pallium, the inner nacreous layer of the shells showed a disordered growth. These results indicated that the obtained Pm-TIMP in this study participated in nacre formation.     

Isolation and functional characterization of DNA damage repair protein (DRT) from Lepidium latifolium L.

We have isolated and in silico characterized a cold regulated plastocyanin encoding gene from Lepidium latifolium L designated as LlaDRT. Its cDNA sequence (JN214346) consists of a 504 bp ORF, 48 and 205 bp of 5′ and 3′ UTR regions, respectively encoding a protein of 17.07 KDa and pI 4.95. In silico and phylogenetic analysis of LlaDRT suggested that the protein has features of a typical plastocyanin family member and of a nearest relative of the predominant isoform of Arabidopsis (PETE2) plastocyanin. Validation of stress response of LlaDRT by qPCR under different abiotic stress regulators viz salicylic acid, jasmonic acid, calcium chloride, ethylene and abscisic acid revealed its possible regulation and crosstalk amongst different pathways.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

PmMGST3, a novel microsomal glutathione S-transferase gene in the dinoflagellate Prorocentrum minimum, is a potential biomarker of oxidative stress

In this study, we evaluated a novel microsomal glutathione S-transferase3 (MGST3) gene from the dinoflagellate Prorocentrum minimum, and examined its expression pattern in response to copper-and nickel-induced stresses. The full length of PmMGST3 was 732 bp, ranging from the dinoflagellate splice leader (DinoSL) sequence to the poly (A) tail, covering a 441-bp ORF, 97-bp 5′UTR, and 194-bp 3′UTR. The PmMGST3 was up-regulated by metals, including copper and nickel. The highest up-regulation levels of the PmMGST3 were found under 0.1 mg/L copper and 0.5 mg/L nickel treatment, respectively. In addition, the PmMGST3 was gradually up-regulated by 0.1 mg/L copper with increasing exposure time. Furthermore, ROS production and reduced GSH was measured in the copper treated cells. A significant increased ROS production and reduced GSH were found in the copper treated cells. These results suggest that PmMGST3 may be related to defense mechanisms associated with oxidative stress in dinoflagellates.     

Plastid trnF pseudogenes are present in Jaltomata, the sister genus of Solanum (Solanaceae): Molecular evolution of tandemly repeated structural mutations

Extensive gene duplication arranged in a tandem array is rare in the plastome of embryophytes. Interestingly, we found pseudogene copies of the trnF gene in the genus Jaltomata, the sister genus of Solanum where such gene duplication has been previously reported. In each Jaltomata sequence available we found two pseudogene copies in close 5′-proximity to the original functional gene. The size of each pseudogene copy ranged between 17 and 48 bp and the anticodon domain was identified as the most conserved element. A common ATT(G)_n motif is particularly interesting and its modifications were found to border the 3′ of the duplicated regions. Other motifs were partial residues, or entire parts of the T- and D-domains, and both domains proved to be variable in length among the pseudogenes identified. The residues of the 3′ and 5′ acceptor stem were not found among the copies. We further compared the newly discovered copies of Jaltomata with those ones previously described from Solanum and inferred phylogenetic relationships of the copies aligned. The evolution of Solanum copies, in contrast to Jaltomata, is hard to explain as resulting only in parsimonious changes since reticulate evolutionary patterns were detected among the copies. The dynamic evolutionary patterns of Solanum might be explained by possible inter- or intrachromosomal recombination.     

The mitochondrial genome of the Russian wheat aphid Diuraphis noxia: Large repetitive sequences between trnE and trnF in aphids

To characterize aphid mitochondrial genome (mitogenome) features, we sequenced the complete mitogenome of the Russian wheat aphid, Diuraphis noxia. The 15,784-bp mitogenome with a high A + T content (84.76%) and strong C skew (− 0.26) was arranged in the same gene order as that of the ancestral insect. Unlike typical insect mitogenomes, D. noxia possessed a large tandem repeat region (644 bp) located between trnE and trnF. Sequencing partial mitogenome of the cotton aphid (Aphis gossypii) further confirmed the presence of the large repeat region in aphids, but with different repeat length and copy number. Another motif (58 bp) tandemly repeated 2.3 times in the control region of D. noxia. All repeat units in D. noxia could be folded into stem-loop secondary structures, which could further promote an increase in copy numbers. Characterization of the D. noxia mitogenome revealed distinct mitogenome architectures, thus advancing our understanding of insect mitogenomic diversities and evolution.     

Polymorphisms of the TLR4 gene and risk of gastric cancer

Objective

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene–environmental interactions on the risk of GC in Northeastern China.

Methods

We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models.

Results

Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR = 0.33, 95% CI 0.18–0.60, P < 0.001 and adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC.

Conclusion

Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC.     

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination

Discrimination of Leishmaniainfantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteineproteaseB (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3’ end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.