Structure of the BoLA‐DRB3 gene and promoter

The cattle major histocompatibility complex (MHC) class II DR gene product is a heterodimer encoded by the BoLA‐DRA and ‐DRB3 genes. Several groups have isolated cDNA and genomic clones for these genes, but their full genomic organization has not been described. We used a combination of long‐range polymerase chain reaction (PCR), cloning and sequencing to define the organization of the DRB3 gene on existing genomic clones and in genomic DNA. We estimate the size of the coding region to be 11.4 kbp. Sequencing of full‐length PCR clones from two different haplotypes confirmed that they carried complete DRB3 genes and allowed the design of probes and primers to isolate and characterize the DRB3 promoter and 3′ end. Fragments carrying the 5′ end of the DRB3 gene and its promoter were identified on bacterial artificial chromosome (BAC) clones carrying the BoLA‐DR genes. A 10‐kbp promoter fragment was subcloned from one clone and a 1.7‐kbp region including exon 1 and the promoter was sequenced. A 3‐kbp fragment encoding exons 4–6 and the entire 3′ untranslated region of the DRB3 gene was isolated from lambda clone A1 and sequenced. This provides us with improved characterization of the DRB3*0101 and DRB3*2002 alleles, and also subcloned 5′ and 3′ flanking regions of the polymorphic DRB3 gene for use in functional studies.     

Sequencing of 15 new BoLA-DRB3 alleles

The class II DR of bovine major histocompatibility complex of cattle (BoLA) plays a central role in the regulation of the immune response through their ability to present those peptides to T-cell receptors. In this work, we sequenced the exon2 of DRB3 to identify new alleles in Chinese yellow cattle, a total of 15 new BoLA-DRB3 alleles were found.     

New polymorphisms for the BoLA-DRB3 upstream regulatory region

Two new alleles, named BoLA-DRB3-P*06 and BoLA-DRB3-P*07, have been identified for the upstream regulatory region of the BoLA-DRB3 gene. The 228-bp nucleotide sequences of the promoter comprising the W, X, Y, CAAT and TATA regulatory boxes were analysed. The BoLA-DRB3-P*06 exhibits one insertion between the W and X boxes, and one transition between the X and Y boxes. On the other hand, the BoLA-DRB3-P*07 showed one insertion in the X box.     

Identification and analysis of the promoter region of the STGC3 gene

Introduction

Nasopharyngeal carcinoma (NPC) is a common malignant tumor of the head and neck. The STGC3 gene is related to development of nasopharyngeal cancer. The aim of this study is to explore the promoter region of the STGC3 gene.

Material and methods

The bioinformatic technique was applied to predict its promoter region and construct the gene promoter region luciferase for the gene vector and transfection of the human embryonic kidney epithelial 293T cell line, human nasopharyngeal carcinoma CNE2 cell line and immortalized nasopharyngeal epithelial NP69 cell line. The recombinant plasmid pGL3-en283, pGL3-en281, pGL3-en571, empty plasmid pGL3-control, negative control pGL3-enhance and internal control of marine intestine luciferase expression vector pRL-SV40 were transfected into NP69 cells, 293T cells and CNE2 cells. Dual luciferase activity detection showed luciferase luminescence values and marine intestine luciferase luminescence values. Relative luciferase activity (RLA) in each cell was calculated.

Results

We observed strong promoter activity of plasmid pGL3-en283, pGL3-en281 and pGL3-en571 in NP69, 293T and CNE2 cells compared with the negative control pGL3-enhance plasmid. Among them, pGL3-en281 showed the strongest promoter activity, and these three kinds of recombinant plasmids showed stronger promoter activity in 293T cells than in CNE2 cells.

Conclusions

The pGL3-en281 plasmid showed stronger promoter activity than pGL3-en571 in the three cells, indicating that –11048 bp to –653 bp might be the core promoter region.     

Establishment of a sequence-based typing system for BoLA-DRB3 exon 2

A rapid, high-resolution sequence-based typing (SBT) system for BoLA-DRB3 exon 2 was developed. Amplification of the entire exon was achieved by a fully nested PCR with locus-specific primers and sequencing was performed directly on the PCR product. Heterozygous sequence data were obtained by automated sequence analysis of both alleles. Forward and reverse sequence data were assembled to improve identification of all heterozygous positions. Specific software (Haplofinder, Roslin Institute Software, Roslin, UK) was designed for allele assignment. Fifty-four females from a Holstein-Charolais resource herd cross, their 12 sires and five unrelated Holstein animals were used to establish the method. In parallel, these animals were typed by DRB3 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to confirm the results. Polymerase chain reaction-RFLP analysis defined 15 known types in the 71 animals, while SBT of the same animals showed 19 known alleles. Subsequently, 72 more animals from the same resource herd were typed by the established SBT method without PCR-RFLP typing. This SBT strategy and the Haplofinder software can be applied to the analysis of any polymorphic locus for which suitable locus-specific primers and allelic sequences are available.     

Association study of promoter polymorphisms within the NOS3 gene and allergic diseases

BACKGROUND: Nitric oxide (NO) is an important mediator of physiologic and pathologic processes in the airways. On this basis, we hypothesized that polymorphisms in the NOS3 gene could be associated with the disease process. METHODS: Two promoter variants (-786C/T and -691C/T) were examined in a Caucasian Czech population of allergic patients [n = 671, with a subgroup of asthmatics (n = 305)] and healthy controls (n = 334) using PCR-RFLP analyses. RESULTS: NOS3 -786C/T and -691C/T were not associated with allergic diseases or asthma. However, the -786 variant was significantly associated with asthma in men (p < 0.01, p(corr) < 0.05) but not in women. NOS3 -691C/T was found to be in strong linkage disequilibrium with -786C/T, and the distribution of combined genotypes was marginally different between the asthmatic and control men. CONCLUSION: Our results suggest that NOS3 gene variants may be one of the factors that participate in the pathogenesis of asthma in men.     

FoxP3 gene promoter polymorphism affects susceptibility to preeclampsia

BackgroundPreeclampsia (PE) is a multifactorial pregnancy disorder and is a major cause of maternal morbidity and mortality. Despite intense study, the pathophysiology of preeclampsia remains enigmatic. Recent studies have reported that regulatory T cells (Tregs) is linked with PE. It is well identified that FoxP3/Scurfin is involved in development and function of Tregs. However, the association between PE and the FoxP3 gene polymorphism has not been sufficiently investigated. In this study, we hypothesized that polymorphisms of the FoxP3 may be related to PE.MethodsWe assessed the relationship between four single-nucleotide polymorphisms (SNPs) in the FoxP3 genes with sequence-specific primers (PCR-SSP) in 81 PE patients and 90 age-matched controls.ResultWe identified significant difference of rs4824747 GG genotype frequency between the PE and control groups. Women with GG genotypes exhibited higher (OR = 6.25, 95% CI = 2.63–14.85; P < 0.0001) risk of developing PE. None of the other investigated SNPs (rs2232365, rs3761547 and rs3761548) showed significant association with PE.ConclusionWe suggest that FoxP3 polymorphisms (rs4824747) could be a potential contributor for the development of PE in Iranian women.     

人肝细胞癌金属蛋白酶组织抑制因子-3的表达及与其基因启动子甲基化的关系

目的 探讨肝细胞癌的发生和门脉浸润机制。方法 20例肝细胞癌患者,每例在手术后分别取原发瘤、门脉瘤栓及远离肝癌之肝组织。用Western印迹法检测金属蛋白酶组织抑制因子(TIMP)的蛋白表达,用逆转录-聚合酶链反应(RT-PCR)检测TIMP-3 mRNA的表达,用甲基化特异性PCR检测TIMP-3基因启动子的甲基化。结果 远离肝癌之肝组织中均可见TIMP-3蛋白和mRNA的表达,原发瘤和门脉瘤栓组织TIMP-3蛋白和mRNA表达明显降低,其中分别有5例和6例完全丢失。远离肝癌之肝组织均未发现TIMP-3启动子甲基化。原发瘤中有7例、门脉瘤栓组织中有9例出现甲基化。所有有甲基化的肝癌组织,包括原发瘤和门脉瘤栓,有13例TIMP-3 mRNA和蛋白表达完全丢失,6例表达降低。TIMP-3启动子甲基化和肝细胞癌组织学分级无关(P>0.05)。结论 肝细胞癌的发生和门脉浸润与TIMP-3基因和蛋白缺失或降低相关、而TIMP-3启动子甲基化是其基因和蛋白缺失或降低的原因。     

Aberrant promoter methylation of the PAQR3 gene is associated with prostate cancer

Methylation markers are promising tools for diagnosis, prognosis and targeted treatment of cancer. In prostate carcinoma, aberrant promoter hypermethylation occurs earlier in the disease course and more consistently than recurrent somatic mutations. PAQR3, a tumor suppressor gene, was recently found to be downregulated in prostate cancer cell lines. We hypothesized that promoter methylation could be responsible for PAQR3 silencing in prostate cancer tissues. We aimed to investigate PAQR3 promoter methylation in prostate cancer by comparing it to benign prostatic hyperplasia (BPH). A total of 154 human prostate tissue samples, including 92 cases with prostate cancer and 62 cases with BPH, were examined by methylation-specific PCR. Clinicopathological correlation between PAQR3 promoter methylation and prognostically relevant variables was studied by statistical analysis. Promoter methylation of PAQR3 was significantly more frequent in prostate carcinoma compared to BPH (73.9% vs. 25.8%, p < 0.01). The high prevalence of PAQR3 methylation in cancer foci was also confirmed with microdissection technique in 12 samples of prostate adenocarcinoma. PAQR3 hypermethylation was associated with perineural invasion (p = 0.03), an adverse clinicopathological feature of prostate cancer. We concluded that PAQR3 can be a promising methylation marker candidate for the detection and monitoring of prostate cancer.     

Novel polymorphisms in the promoter region of the neurotrophin-3 gene and their associations with schizophrenia

Based on the neurodevelopmental hypothesis in the etiology of schizophrenia, neurotrophic factors may be involved in the pathogenesis of the illness. We searched for polymorphisms in the promoter region of the neurotrophin-3 (NTF3) gene by using denaturing high performance liquid chromatography (DHPLC). Six single nucleotide polymorphisms (SNPs) were found. When these polymorphisms were examined for association with schizophrenia, a weakly significant difference was observed in the genotype distribution of the G/- 3004/A polymorphism between 184 schizophrenics and 185 controls (P < 0.05), although no statistically significant association was detected in a family-based sample of 50 trios (schizophrenics and their parents). With respect to the other polymorphisms, there was no significant association with schizophrenia. The G/- 3004/A polymorphism was in linkage disequilibrium with the CA repeat polymorphism in the first intron of the NTF3 gene. When haplotype-based analysis was performed, an increased frequency of the haplotype containing the G(- 3004) and the "A3" ([CA]23) alleles was observed for the schizophrenics compared to controls. Our results suggest that the G(- 3004)-A3 haplotype has a modest effect of giving susceptibility to schizophrenia.     

Association analysis of GRK3 gene promoter variants in cocaine abuse

The G protein-coupled receptor kinase 3 gene (GRK3) is a candidate gene for cocaine addiction because it is involved in the regulation of several neurotransmitter receptors, including the response to dopaminergic agonists such as methamphetamine and cocaine. We hypothesized that genetic variants in the GRK3 gene might be associated with an increased risk of cocaine addiction. To test this, we genotyped three variants located in 5' untranslated and promoter regions of the gene in a sample of 711 cocaine users and 862 healthy control individuals from Sao Paulo, Brazil. Genotypic, allelic and haplotypic analyses provided no evidence for an association between alleles at these polymorphisms and cocaine abuse in this sample. Population stratification was tested for and its effect corrected for, but this did not affect the association test results. In conclusion, our results do not support a major role for GRK3 gene promoter variants in cocaine addiction.     

RUNX3基因启动子甲基化与胃癌关系的meta分析

目的采用meta分析的方法评价RUNX3基因启动子甲基化与胃癌发生的关系。方法检索公开发表在Pubmed、EMBASE、Ovid、中国期刊全文数据库（CNKI）关于RUNX3基因启动子甲基化与胃癌发生关系的研究。应用Statall．0统计软件分析RUNX3基因启动子甲基化与胃癌发生间的关联。结果共有19项研究纳入分析．meta分析结果显示：胃癌患者癌组织中RUNX3基因启动子甲基化发生率为P=55％（95％CI：45％～66％）：胃癌患者远癌正常胃组织中RUNX3基因启动子甲基化发生率为P=18％（95％CI：7％～29％）。与远癌正常胃组织相比,胃癌组织中RUNX3基因启动子甲基化发生优势比OR=7．85（95％CI：5．81～10．61）。结论胃癌患者癌组织中RUNX3基因启动子甲基化发生率明显高于远癌正常胃组织,RUNX3基因启动子甲基化可能与胃癌的发生密切相关。     

Identification of four novel alleles of the BoLA-DRB3 upstream regulatory region in Chinese yellow cattle

The sequence of upstream regulatory region (URR) of BoLA-DRB3 gene was amplified with polymerase chain reaction followed by DNA sequencing from six animals of Chinese yellow cattle. A total of five alleles including four newly identified ones, named BoLA-DRB3*R-03-U2, BoLA-DRB3*R-06-U2, BoLA-DRB3*R-07-U and BoLA-DRB3*R-12-U for the BoLA-DRB3 URR were found. Result of sequence analysis showed that the regulatory elements W, X, Y, CCAAT and TATA-like boxes existed in such URRs and 16 polymorphic sites (11 transitions, 3 transversions, 1 deletion and 1 insertion) located in the spacers between the conserved consensus boxes and 1 insertion within X box, while no new polymorphic site within the consensus boxes.