Mutations in pre-core and basic core promoter regions of hepatitis B virus in chronic hepatitis B patients

AIM: To investigate the frequency of mutations in pre-core (pre-C) and basic core promoter (BCP) regions of hepatitis B virus (HBV) from Shanxi Province, and the association between mutations and disease related indexes.METHODS: One hundred chronic hepatitis B patients treated at Shanxi Province Hospital of Traditional Chinese Medicine were included in this study. PCR-reverse dot blot hybridization and mismatch amplification mutation assay (MAMA)-PCR were used to detect the mutations in the HBV pre-C and BCP regions. HBV DNA content and liver function were compared between patients with mutant HBV pre-C and BCP loci and those with wild-type loci. The consistency between PCR-reverse dot blot hybridization and MAMA-PCR for detecting mutations in the HBV pre-C and BCP regions was assessed.RESULTS: Of the 100 serum samples detected, 9.38% had single mutations in the pre-C region, 29.17% had single mutations in the BCP region, 41.67% had mutations in both BCP and pre-C regions, and 19.79% had wild-type loci. The rates of BCP and pre-C mutations were 65.7% and 34.3%, respectively, in hepatitis B e antigen (HBeAg) positive patients, and 84.6% and 96.2%, respectively, in HBeAg negative patients. The rate of pre-C mutations was significantly higher in HBeAg negative patients than in HBeAg positive patients (χ2 = 26.62, P = 0.00), but there was no significant difference in the distribution of mutations in the BCP region between HBeAg positive and negative patients (χ2 = 2.43, P = 0.12). The presence of mutations in the pre-C (Wilcoxon W = 1802.5, P = 0.00) and BCP regions (Wilcoxon W = 2906.5, P = 0.00) was more common in patients with low HBV DNA content. Both AST and GGT were significantly higher in patients with mutant pre-C and BCP loci than in those with wild-type loci (P < 0.05). PCR-reverse dot blot hybridization and MAMA-PCR for detection of mutations in the BCP and pre-C regions had good consistency, and the Kappa values obtained were 0.91 and 0.58, respectively.CONCLUSION: HBeAg negative patients tend to have HBV pre-C mutations. However, these mutations do not cause increased DNA copies, but associate with damage of liver function.

Analysis of SNPs in the Promoter Region of the Growth Hormone(GH) Gene in Mini-pigs

Single nucleotide polymorphisms(SNPs) of the growth hormone(GH) gene were investigated in six pig breeds,consisting of four mini-pig breeds(Wuzhishan,Bama,Xiang and Tibet pig),and two others(Dahlan and Landrace pig).Three pairs of primers for promoter regions of the GH gene were designed on the basis of the pig genomic sequence and SNPs were detected by the PCR-SSCP method.The results indicated three mutations in the 5’-flanking region.The analysis results showed that the frequencies of allele A and D in four mini-pig breeds were higher than that in other breeds at a locus within the 5’-flanking region(P【0.05).These results suggest that differences in body size may be associated with these SNPs of 5’-flanking region and amino acid mutation of the signal peptide of GH in these pig breeds.

Cloning and Analysis of the Promoter Region of Rat uPA Gene

Objective To clone and analyze the promoter sequence of rat urokinase plasminogen activator protein gene. Methods The genomic DNA was extracted from rat testicular tissue. According to urokinase plasminogen activator, the gene sense primer and antisense primer of uPA gene were designed and synthesized, then Touch-Down PCR were performed. After proper purification, the PCR product was sequenced, analyzed with the promoter prediction software and compared with the DNA sequence of rattuas urokinase plasminogen activator. Results The cloned uPA gene was about 1 572 bp in length, which contained a full open-reading frame with 21 bp in length exons, and the upper region of transcriptional start was 1 551 bp in length which was eucaryon transcriptional control area. The 5＇ UTR had a promoter region including a non-responsive TATA-box. Not only the GC-box binding region was found in this gene, but also active protein I （AP1） and SP1 were seen in other regions. Conclusion A 1 572 bp uPA gene fragment （GenBank accession No.X65651） was obtained from rat genomic DNA library, containing eucaryon transcriptional control area with a promoter region, non-conspicuous TATA-box, GC-box and an extron. A non-responsive TATA-box is located at the upper -30 region.

Association of Genetic Variation in the Promoter Region of OXTR with Differences in Social Affective Neural Processing

Evidence supports the involvement of oxytocin in social behavior. The oxytocin receptor gene (OXTR) has been associated with differences in social brain function and risk for autism. Motivated by recent work, we investigated the effect of variation in the common functional rs2268498 T/C polymorphism in the promoter region of OXTR on neural responses to fear expressions. 46 healthy subjects were divided into genotype groups of C carriers (n = 32) and TT ho-mozygous (n = 14) and neural activity was measured during the recognition of fear and neutral expressions. Results showed that during the recognition of fear expressions, the TT genotype group exhibited increased responding in the inferior occipital gyrus, considered important for face processing, compared to carriers of the C allele (P < 0.005;cluster corrected for whole brain), an effect not found for neutral faces. These results indicate the impact of this OXTR genetic variant on individual differences in social affective neural processing.

Study on the Promotion of Digital Trade to Northeast Asian Regional Economy

Society is currently transitioning from the era of informatization and digitization to the era of intelligence.Advanced technologies such as artificial intelligence,the Internet of Things,and big data are permeating every aspect of production and life.This penetration presents a rare opportunity for the development of digital trade and catalyzes innovation in traditional trade patterns.This paper first analyzes the inevitability of digital trade development in Northeast Asia.Secondly,it delves into the challenges faced by digital trade development and explores in-depth the promotion strategy for digital trade’s impact on the economic development of the Northeast Asia region,providing insights from various perspectives for reference.

EVOLUTIONARY FUZZY GUIDANCE LAW WITH SELF-ADAPTIVE REGION

Effective guidance is one of the most important tasks to the performance of air-to-air missile. The fuzzy logic controller is able to perform effectively even in situations where the information about the plant is inaccurate and the operating conditions are uncertain. Based on the proportional navigation, the fuzzy logic and the genetic algorithm are combined to develop an evolutionary fuzzy navigation law with self-adapt region for the air-to-air missile guidance. The line of sight (LOS) rate and the closing speed between the missile and the target are inputs of the fuzzy controller. The output of the fuzzy controller is the commanded acceleration. Then a nonlinear function based on the conventional fuzzy logic control is imported to change the region. This nonlinear function can be changed with the input variables. So the dynamic change of the fuzzy variable region is achieved. The guidance law is optimized by the genetic algorithm. Simulation results of air-to-air missile attack using MATLAB show that the method needs less acceleration and shorter flying time, and its realization is simple.[KH*3/4D]

牙鲆GHR基因Promoter区微卫星序列多态性与生长性状关系的初步研究

采用牙鲆GHR基因5’端Promoter区的1个微卫星标记,对胶南和日照2个牙鲆养殖群体进行了群体遗传多样性的研究,并探索该基因多态性位点与牙鲆生长性状之间的相关性。结果表明,2个群体在该座位的等位基因数为12和9个,有效等位基因数为6.26和5.04个,多态信息含量为0.84和0.80。2个群体该座位的Hardy-Weinberg遗传偏离指数均为正值,并没有显示出杂合子缺失,但各基因型分布频率都在一定程度上偏离Hardy-Weinberg平衡(P<0.01)。连锁分析中发现,在胶南群体中,IM基因型对应的个体在全重、全长、体长、头长、体高和眼径形态学数据中均是最大的;在日照群体中,BC基因型对应的个体在全重、全长、体高、尾柄高、尾柄长和眼径数据中均是最大的;而CJ基因型对应的个体在体长和头长这两组数据中是最大的。该结果为研究GHR基因的变异对鱼类生长发育的影响及探索将该基因作为生长遗传标记的可行性奠定了实验基础。

GFAP promoter directs lacZ expression specifically in a rat hepatic stellate cell line

AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.

Interleukin-10 promoter polymorphisms in patients with hepatitis B virus infection or hepatocellular carcinoma in Chinese Han ethnic population

BACKGROUND: Since single nucleotide polymorphisms (SNPs) can serve as gene markers, polymorphism profiles may help scientists to identify the full collection of genes that contribute to the development of complex diseases such as cancer. The distribution of interleukin-10 (IL-10) promoter polymorphisms in Chinese Han ethnic patients with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC) was investigated in this study. METHODS: The polymorphisms of IL-10 promoter region were detected by pulymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing. Sixty-six health controls, 42 patients with HBV infection, 30 HCC patients, and cell line SMMC-7721 were examined this way. RESULTS: Polyrnorphisms of T/C or T/N on-872 site occurred frequently in Han ethnic population. Pulyrnorphisms were detected in HBV and HCC patients and cell line SMMC-7721. The hotspot among the pulymorphisms was inserting base A between-1058 and-1057. CONCLUSION: Polymorphisms of IL-10 promoter in HBV and HCC patients may be associated with HBV infection and HCC development.

Shallow Genetic Structure of Pholis fangi in Bohai Sea and Yellow Sea Inferred from mt DNA Control Region

Pholis fangi is an ecologically important fish species inhabiting Chinese coastal waters of Yellow Sea and Bohai Sea. To investigate the genetic diversity and population genetic structure of P. fangi, a fragment of 487 bp in the first hypervariable region (HVR-1) of the mitochondrial DNA (mtDNA) control region was sequenced for 181 individuals collected from Bohai Sea and Yel- low Sea. A total of 18 polymorphic sites were detected, which defined 25 haplotypes. A moderate level of haplotype diversity (h = 0.7052) and a low level of nucleotide diversity (π= 0.0028) were detected. Both the phylogenetic tree and the haplotype network showed no significant genealogical structure difference among sampling locations. Pairwise FST comparison and hierarchical mo- lecular variance analysis (AMOVA) revealed that no significant genetic structure difference existed throughout the investigated re- gions, suggesting a high gene exchange among different populations. The results of neutrality test and mismatch distribution analysis indicated that a late Pleistocene population expansion (38000 127000 years ago) happened. Seasonal schooling migration may con- tribute to the genetically homogeneous population structure of the species.

Satellite Constellation Design with Multi-Objective Genetic Algorithm for Regional Terrestrial Satellite Network

Constellations design for regional terrestrial-satellite network can strengthen the coverage for incomplete terrestrial cellular network. In this paper, a regional satellite constellation design scheme with multiple feature points and multiple optimization indicators is proposed by comprehensively considering multi-objective optimization and genetic algorithm, and "the Belt and Road" model is presented in the way of dividing over 70 nations into three regular target areas. Following this, we formulate the optimization model and devise a multi-objective genetic algorithm suited for the regional area with the coverage rate under simulating, computing and determining. Meanwhile, the total number of satellites in the constellation is reduced by calculating the ratio of actual coverage of a single-orbit constellation and the area of targets. Moreover, the constellations' performances of the proposed scheme are investigated with the connection of C++ and Satellite Tool Kit(STK). Simulation results show that the designed satellite constellations can achieve a good coverage of the target areas.

Expression Pattern of the Foreign Gene Regulated by the Tomato rbcS3A Promoter in Transgenic Tomato

[Objective] This study aimed to reveal the expression pattern of foreign genes regulated by tomato rbcS3A promoter in transgenic tomato. [Method] Rubisco small subunit promoter rbcS3A was cloned by PCR, fused to the upstream of Gus coding region in a binary vector, and transformed into tomato plants mediated by Agrobacterium. Histochemical staining on PCR positive plants was performed to ana- lyze the expression pattern of the foreign gene regulated by the tomato rbcS3A pro- moter in transgenic tomato. [Result] A total of 15 positive plants were obtained, ac- counting for 33.3%. Histochemical staining showed that the expression level of Gus fusion gene was highest in mature leaf, lower in reproductive organs such as fruit, and not detected in seed. [Conclusion] More positive seedlings were obtained using the modified tissue culture method. Under the control of tomato rbcS3A promoter, exogenous gene highly expressed in transgenic plant leaves, but did not express in seeds and tomato pulp.

Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity

Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

A Method for Upscaling Genetic Parameters of CERES-Rice in Regional Applications

To upscale the genetic parameters of CERES-Rice in regional applications, Jiangsu Province, the second largest rice producing province in China, was taken as an example. The province was divided into four rice regions with different rice variety types, and five to six sites in each region were selected. Then the eight genetic parameters of CERES-Rice, particularly the four parameters related to the yield, were modified and validated using the Trial and Error Method and the local statistical data of rice yield at a county level from 2001 to 2004, combined with the regional experiments of rice varieties in the province as well as the local meteorological and soil data （Method 1）. The simulated results of Method 1 were compared with those of other three traditional methods upscaling the genetic parameters, i.e., using one-site experimental data from a local representative rice variety （Method 2）, using local long-term rice yield data at a county level after deducting the trend yield due to progress of science and technology （Method 3）, and using rice yield data at a super scale, such as provincial, ecological zone, country or continent levels （Method 4）. The results showed that the best fitness was obtained by using the Method 1. The coefficients of correlation between the simulated yield and the statistical yield in the Method 1 were significant at 0.05 or 0.01 levels and the root mean squared error （RMSE） values were less than 9% for all the four rice regions. The method for upscaling the genetic parameters of CERES-Rice presented is not only valuable for the impact studies of climate change, but also favorable to provide a methodology for reference in crop model applications to the other regional studies.

Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F StrainVP1 Protein

Enterovirus 71 （EV71） is a member of the Entero-virus genus of the Picomaviridae family and is the major cause of Hand, foot, and mouth disease （HFMD） in children. Different strains from Gansu were cloned and the P1 protein was sequenced and analysed. Results indicate that there are three kinds of EV71 infections prevalent in Gansu. The VP 1 protein from one of these strains, 55F, was expressed. The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody, the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay. These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.

Mitochondrial DNA control region diversity and population structure of Pacific herring(Clupea pallasii)in the Yellow Sea and the Sea of Japan

To investigate the genetic variation and population structure of Pacific herring in the Yellow Sea and the genetic differentiation between the Yellow Sea and the Sea of Japan, fragments of 479-bp mitochondrial DNA control region were sequenced for 110 individuals collected from three different periods in the Yellow Sea and one locality in the Sea of Japan. High haplotype diversity and moderate nucleotide diversity were observed in Pacific herring. AMOVA and exact test of population differentiation showed no significant genetic differentiations among the three populations of the Yellow Sea and suggested the populations can be treated as a single panmictic stock in the Yellow Sea. However, a large and significant genetic differentiation （ФST=0.11; P=0.00） was detected between the populations in the Yellow Sea and the Sea of Japan. The high sea water temperature in the Tsushima Strait was thought a barrier to block the gene exchange between populations of the two sea areas. The neutrality tests and mismatch distribution indicated recent population expansion in Pacific herring.

Integrated Robust Design Method Based on Maximum Tolerance Region

Traditionally,parameter design is carried out prior to tolerance design. However, this two-step design strategy cannot guarantee optimal robustness for products' quality. The proposed integrated robust design method determined the optimal parameter and tolerance simultaneously by calculating the maximum tolerance region,thereby improving the quality of products. In addition,the proposed method did not need uncertainty analysis to obtain the maximum tolerance region,so that the calculation cost could be decreased. And the method avoided the difficulty of gaining costtolerance function as maximum tolerance region represented both demand of cost and robust. Finally,an amplifier circuit case was conducted for verification purpose. Based on the results, the proposed approach could provide robust solution with optimal maximum tolerance region.

Studies on Genetic Transformation of Fresh-cut Chrysanthemum Using DREB1A Promoted by Stress-induced Promoter rd29A

In this study, DERB1A transcription factor and stress-induced promoter rd29A were isolated respectively and amplified from Arabidopsis thaliana, se- quenced and analyzed by DNAsis. In addition, the stress-induced promoter rd29A was utilized to construct the plant expression vector of DERB1A, which was transformed into Agrobacterium tumefaciens. Furthermore, the transgenic regeneration system of fresh-cut chrysanthemum from callus to plantlets was established successfully. On this basis, chrysanthemum leaf-disc explants were genetically transformed with Agrobacterium-mediated method. Two positive transgenie plantlets were obtained in vitro. Based on PCR detection, DREB1A transcription factor was integrated into chrysanthemum genome, which laid the foundation for breeding new transgenie cultivars of fresh-cut chrysanthemum with high comprehensive stress resistance, good cmalitv and high field.

Genomic structure analysis of SNC6, a progesterone-receptor associated protein gene, and cloning and characterization of its 5＇-flanking region

Objective: To analyze the genomic structure of SNC6, a progesterone\|receptor associated protein gene and its regulatory elements in its 5'\|flanking region. Methods: Genomic sequence from GenBank database (accession number: Z98048) covering the whole SNC6 gene was used to analyze the genomic structure of SNC6 and design primers for PCR amplification of its 5'\|flanking region. A 1894 bp fragment of the 5'\|flanking region \{(-1814\} to +75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments (1423 bp, 632 bp and 416 bp, which correspond to -1344 to +75, -552 to +75 and -337 to +75 respectively), were subcloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luciferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All transfected SW620 cells with the above 5\|flanking region\|containing constructs showed luciferase activities. The highest luciferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luciferase activities were measured in transfected cells with vectors containing 416 bp fragments. Luciferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in transfected cells with vectors containing 1423 bp fragments. Conclusion: The basic transcription\|promoting element (promoter) for SNC6 expression resides between 0 to -337, and two transcription\|enhancing elements (enhancer) resides between -337 to -552 and -1344 to -1814, whereas one transcription\|inhibiting element (silencer) exists between -552 to -1344.

A novel constitutive promoter and its downstream 5′ UTR derived from cotton(Gossypium spp.) drive high-level gene expression in stem and leaf tissues

The development of genetically modified crops requires new promoters and regulatory regions to achieve high gene ex- pression and/or tissue-specific expression patterns in plants. To obtain promoter sequences of plants with new properties, we analyzed the expression traits of the cotton （Gossypium hirsutum） translation elongation factor 1A gene family. The results showed that the GhEF1A8 gene is highly expressed in different organs of cotton plants, and showed much higher transcript levels in stems and leaves. Its promoter （GhEFIA1.7） and the 5＂ untranslated region （5＂ UTR）, comprising a regulatory region named PGhEFIA8, were isolated from cotton and studied in stably transformed tobacco plants. The regulatory region sequences were fused to the 13-glucuronidase （GUS） reporter gene to characterize its expression pattern in tobacco. Histochemical and fiuorometric GUS activity assays demonstrated that PGhEF1A8 could direct GUS gene expression in all tissues and organs in transgenic tobacco, including leaves, stems, flowers, and roots. The level of GUS activity in the leaves and stems was significantly higher than in cauliflower mosaic virus （CaMV） 35S promoter：：GUS plants, but as same as CaMV 35S promoter：：GUS plants in flower and root tissues. GUS expression levels decreased 2-10-fold when the 5＂ UTR was absent from PGhEF1A8. Deletion analysis of the PGhEFIA8 sequence showed that the region -647 to -323 might possess negative elements that repress transgene expression in tobacco plants. The results suggested that the GhEFIA8 regulation region may represent a practical choice to direct high-level constitutive expression of transgenes and could be a valuable new tool in plant genetic engineering.