A Novel Complex A/C/G Intergenotypic Recombinant of Hepatitis B Virus Isolated in Southern China

Hepatitis B virus (HBV) genotypes and subgenotypes may vary in geographical distribution and virological features. Previous investigations, including ours, showed that HBV genotypes B and C were respectively predominant in South and North China, while genotypes A and D were infrequently detected and genotype G was not found. In this study, a novel A/C/G intergenotype was identified in patients with chronic HBV infection in Guilin, a city in southern China. Initial phylogenetic analysis based on the S gene suggested the HBV recombinant to be genotype G. However, extended genotyping based on the entire HBV genome indicated it to be an A/C/G intergenotype with a closer relation to genotype C. Breakpoint analysis using the SIMPLOT program revealed that the recombinant had a recombination with a arrangement of genotypes A, G, A and C fragments. Compared with the HBV recombinants harboring one or two genotype G fragments found in Asian countries, this Guilin recombinant was highly similar to the Vietnam (98–99%) and Long An recombinants (96–99%), but had a relatively low similarity to the Thailand one (89%). Unlike those with the typical genotype G of HBV, the patients with the Guilin recombinant were seropositive for HBeAg. Moreover, a relatively high HBV DNA viral load (>2×10⁶ IU/ml) was detected in the patients, and the analysis of viral replication capacity showed that the Guilin recombinant strains had a competent replication capacity similar to genotypes B and C strains. These findings can aid in not only the clarification of the phylogenetic origin of the HBV recombinants with the genotype G fragment found in Asian countries, but also the understanding of the virological properties of these complicated HBV recombinants.     

含抑制素重组质粒的减毒猪霍乱沙门氏菌C500遗传稳定性研究

为了分析研究含抑制素重组质粒pVAX-IS-asd(以下简写为pXAIS)的crp(cAMP受体蛋白)和asd(天冬氨酸β-半乳搪脱氢酶)基因双缺失猪霍乱沙门氏菌C500菌株的遗传稳定性.方法:将现有的无抗性抑制素重组表达质粒pXAIS转化入crp和asd基因双缺失猪霍乱沙门氏菌C500菌株(简称"空质粒株")中,得"含质粒株".将"含质粒株"细菌在无选择压力条件下传代培养50代.利用酶切鉴定方法,分析"含质粒株"细菌中抑制素pXA/S质粒的稳定性;利用PCR方法扩增菌株("含质粒株"和"空质粒株")保守序列中invA基因,分析各代细菌的invA基因稳定性;通过比较传代培养后各代细菌的生长图型,分析"含质粒株"细菌传代后的生长规律.结果表明,"含质粒株"细菌连续传代50次后,仍能检出pXAIS质粒和invA基因,而且生长规律与"空质粒株"没有明显差异.说明含pXAIS质粒的猪霍乱沙门氏菌crp和asd基因双缺失株具有良好的遗传稳定性.     

Developing a Novel Gene-Delivery Vector System Using the Recombinant Fusion Protein of Pseudomonas Exotoxin A and Hyperthermophilic Archaeal Histone HPhA

Non-viral gene delivery system with many advantages has a great potential for the future of gene therapy. One inherent obstacle of such approach is the uptake by endocytosis into vesicular compartments. Receptor-mediated gene delivery method holds promise to overcome this obstacle. In this study, we developed a receptor-mediated gene delivery system based on a combination of the Pseudomonas exotoxin A (PE), which has a receptor binding and membrane translocation domain, and the hyperthermophilic archaeal histone (HPhA), which has the DNA binding ability. First, we constructed and expressed the rPE-HPhA fusion protein. We then examined the cytotoxicity and the DNA binding ability of rPE-HPhA. We further assessed the efficiency of transfection of the pEGF-C1 plasmid DNA to CHO cells by the rPE-HPhA system, in comparison to the cationic liposome method. The results showed that the transfection efficiency of rPE-HPhA was higher than that of cationic liposomes. In addition, the rPE-HPhA gene delivery system is non-specific to DNA sequence, topology or targeted cell type. Thus, the rPE-HPhA system can be used for delivering genes of interest into mammalian cells and has great potential to be applied for gene therapy.     

Genetic Differences among the a/J x C57bl/6j Recombinant Inbred Mouse Lines and Their Degree of Association with Glucocorticoid-Induced Cleft Palate

Hydrocortisone sodium phosphate was injected intramuscularly into A/J, C57BL/6J and recombinant inbred lines from these two parental lines to study the genetics of steroid-induced cleft palate in a situation of identical maternal and fetal genotypes. The strains were typed for H-2 (the major histocompatibility locus), beta-glucuronidase and beta 2-microglobulin, which served as markers on chromosomes 17, 5 and 2, respectively. Hepatic glucocorticoid binding capacity had been previously measured in Hepes buffer and Hepes buffer plus dithiothreitol (DTT). The level of glucocorticoid binding in Hepes buffer and in Hepes plus DTT, as well as their ratio, was compared to the incidence of steroid-induced cleft palate in the recombinant inbred lines. A correlation was found between the response of glucocorticoid binding to DTT (expressed as a ratio of binding in the presence of DTT to binding without DTT) and hydrocortisone-induced cleft palate. When analyzing the effect of the three chromosomal markers on hydrocortisone-induced cleft palate, the b alleles of beta 2-microglobulin and of beta-glucuronidase were associated with a higher incidence. Genetic analyses of the differences between these two inbred strains of mice in the incidence of steroid-induced cleft palate show it not to be monogenic.     

Rapid Evolution of Lifespan in a Novel Environment: Sex-Specific Responses and Underlying Genetic Architecture

Animal lifespans can vary substantially among closely related species and even among conspecific populations, but it is often difficult to identify environmental and genetic factors producing such variation. We used experimental evolution to examine how transfer to a novel environment affects adult lifespan and rates of senescence in a seed-feeding beetle. Three replicate lines of Callosobruchus maculatus (F.) were switched to a new host plant (cowpea), and each evolved shorter adult lifespans compared to a line maintained on the ancestral host (mung bean). However, the evolution of lifespan differed between the sexes; female lifespan was reduced by ~11% in all cowpea replicates, whereas male lifespan decreased by an average of only 5.6% and the magnitude of the reduction varied among replicates. Reduced lifespan in lines switched to cowpea mirrored the shorter lifespan observed in a separate population chronically associated with cowpea. We then performed crosses between the mung bean and cowpea lines to estimate the genetic architecture underlying the rapid evolution of a shorter lifespan on cowpea. Dominance (overdominance) contributed substantially to the difference between the cowpea and mung bean lines for female lifespan but not for male lifespan. However, details of the genetic architecture varied among the three replicate crosses, so that the convergent evolution of shorter female lifespan in the different cowpea lines did not arise from identical allelic substitutions. Our study demonstrates that insect lifespan can be predictably modified by a switch to a novel host plant, that both the magnitude of this response and its underlying genetic architecture can be sex-specific, and that convergent evolution of a complex trait such as lifespan can arise from different genetic mechanisms.     

A Novel Analytical Framework for Dissecting the Genetic Architecture of Behavioral Symptoms in Neuropsychiatric Disorders

Background

For diagnosis of neuropsychiatric disorders, a categorical classification system is often utilized as a simple way for conceptualizing an often complex clinical picture. This approach provides an unsatisfactory model of mental illness, since in practice patients do not conform to these prototypical diagnostic categories. Family studies show notable familial co-aggregation between schizophrenia and bipolar illness and between schizoaffective disorders and both bipolar disorder and schizophrenia, revealing that mental illness does not conform to such categorical models and is likely to follow a continuum encompassing a spectrum of behavioral symptoms.

Results and Methodology

We introduce an analytic framework to dissect the phenotypic heterogeneity present in complex psychiatric disorders based on the conceptual paradigm of a continuum of psychosis. The approach identifies subgroups of behavioral symptoms that are likely to be phenotypically and genetically homogenous. We have evaluated this approach through analysis of simulated data with simulated behavioral traits and predisposing genetic factors. We also apply this approach to a psychiatric dataset of a genome scan for schizophrenia for which extensive behavioral information was collected for each individual patient and their families. With this approach, we identified significant evidence for linkage among depressed individuals with two distinct symptom profiles, that is individuals with sleep disturbance symptoms with linkage on chromosome 2q13 and also a mutually exclusive group of individuals with symptoms of concentration problems with linkage on chromosome 2q35. In addition we identified a subset of individuals with schizophrenia defined by language disturbances with linkage to chromosome 2p25.1 and a group of patients with a phenotype intermediate between those of schizophrenia and schizoaffective disorder with linkage to chromosome 2p21.

Conclusions

The findings presented are novel and demonstrate the efficacy of this approach in detection of genes underlying such complex human disorders as schizophrenia and depression.     

Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:CC:G (28 cases) and G:CT:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5-PuGA-3 was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5PyGN3. G:CT:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:CC:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

A Discrete Event Simulation Model for Evaluating the Performances of an M/G/C/C State Dependent Queuing System

M/G/C/C state dependent queuing networks consider service rates as a function of the number of residing entities (e.g., pedestrians, vehicles, and products). However, modeling such dynamic rates is not supported in modern Discrete Simulation System (DES) software. We designed an approach to cater this limitation and used it to construct the M/G/C/C state-dependent queuing model in Arena software. Using the model, we have evaluated and analyzed the impacts of various arrival rates to the throughput, the blocking probability, the expected service time and the expected number of entities in a complex network topology. Results indicated that there is a range of arrival rates for each network where the simulation results fluctuate drastically across replications and this causes the simulation results and analytical results exhibit discrepancies. Detail results that show how tally the simulation results and the analytical results in both abstract and graphical forms and some scientific justifications for these have been documented and discussed.     

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV^901_09 and rISAVr^S6-NotI-HPR containing a NotI restriction site and rISAV^S6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 10⁵ PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.     

汉族人群NOS3 A-922G、NOS3 T-786C与NOS3 G894T SNP的等位基因及其组合分布与高血压的相关性

为了分析汉族人群一氧化氮合酶基因NOS3 A-922G、NOS3 T-786C与NOS3 G894T单核苷酸多态性(sin-gle nucleotide polymorphism,SNP)的等位基因及其组合分布与高血压病的相关性,选取无亲缘关系的高血压病人192例(男97例,女95例)以及无亲缘关系的健康个体122例(男76例,女46例)为对照组,提取静脉血白细胞基因组DNA,采用等位基因特异性引物PCR技术检测NOS3 A-922G、NOS3 T-786C与NOS3 G894T 3个位点的基因型.其结果显示:高血压病组与对照组NOS3 G894T、NOS3A-922G及NOS3 T-786C各等位基因型及其基因单倍型频率比较无显著性差异(P＞0.05).男、女性别分层研究:无论男亚组还是女亚组均未发现NOS3 A-922G、NOS3T-786C与NOS3 G894T各个位点SNP与高血压病有相关性.等位基因组合分布研究发现NOS3 G894G A-922G T-786T组合基因型总体频率分布在高血压病组与正常对照组之间有显著性差异(P＜0.05,x2=4.5944).男、女性别分层研究:男亚组上述3个位点SNP的各个组合基因型分布频率在高血压病组与正常对照组之间无显著性差异(P＞0.05);女亚组中携带NOS3 G894G A-922G T-786C的组合基因型分布频率在高血压病组与正常对照组之间有显著性差异(P＜0.01,x2=8.502).研究发现,在中国汉族人群中NOS3A-922 G、NOS3 T-786C与NOS3G894T SNP与高血压病无明确的相关性,且无性别差异.组合分布研究发现,NOS3 G894G A-922G T-786C的组合基因型分布频率在高血压病女性亚组较健康女性亚组明显减低,提示携带该组合基因型女性人群可能不易患高血压病.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.     

Novel Tools for Genetic Manipulation of Follicle Stem Cells in the Drosophila Ovary Reveal an Integrin-Dependent Transition from Quiescence to Proliferation

In many tissues, the presence of stem cells is inferred by the capacity of the tissue to maintain homeostasis and undergo repair after injury. Isolation of self-renewing cells with the ability to generate the full array of cells within a given tissue strongly supports this idea, but the identification and genetic manipulation of individual stem cells within their niche remain a challenge. Here we present novel methods for marking and genetically altering epithelial follicle stem cells (FSCs) within the Drosophila ovary. Using these new tools, we define a sequential multistep process that comprises transitioning of FSCs from quiescence to proliferation. We further demonstrate that integrins are cell-autonomously required within FSCs to provide directional signals that are necessary at each step of this process. These methods may be used to define precise roles for specific genes in the sequential events that occur during FSC division after a period of quiescence.     

Recombinant human Metapneumovirus lacking the small hydrophobic SH and/or attachment G glycoprotein: deletion of G yields a promising vaccine candidate

Human metapneumovirus (HMPV) has recently been identified as a significant cause of serious respiratory tract disease in humans. In particular, the emerging information on the contribution of HMPV to pediatric respiratory tract disease suggests that it will be important to develop a vaccine against this virus for use in conjunction with those being developed for human respiratory syncytial virus and the human parainfluenza viruses. A recently described reverse genetic system (S. Biacchesi, M. H. Skiadopoulos, K. C. Tran, B. R. Murphy, P. L. Collins, and U. J. Buchholz, Virology 321:247-259, 2004) was used to generate recombinant HMPVs (rHMPVs) that lack the G gene, the SH gene, or both. The DeltaSH, DeltaG, and DeltaSH/G deletion mutants were readily recovered and were found to replicate efficiently during multicycle growth in cell culture. Thus, the SH and G proteins are not essential for growth in cell culture. Apart from the absence of the deleted protein(s), the virions produced by the gene deletion mutants were similar by protein yield and gel electrophoresis protein profile to wild-type HMPV. When administered intranasally to hamsters, the DeltaG and DeltaSH/G mutants replicated in both the upper and lower respiratory tracts, showing that HMPV containing F as the sole viral surface protein is competent for replication in vivo. However, both viruses were at least 40-fold and 600-fold restricted in replication in the lower and upper respiratory tract, respectively, compared to wild-type rHMPV. They also induced high titers of HMPV-neutralizing serum antibodies and conferred complete protection against replication of wild-type HMPV challenge virus in the lungs. Surprisingly, G is dispensable for protection, and the DeltaG and DeltaSH/G viruses represent promising vaccine candidates. In contrast, DeltaSH replicated somewhat more efficiently in hamster lungs compared to wild-type rHMPV (20-fold increase on day 5 postinfection). This indicates that SH is completely dispensable in vivo and that its deletion does not confer an attenuating effect, at least in this rodent model.     

Genetic Determinants of the Network of Primary Metabolism and Their Relationships to Plant Performance in a Maize Recombinant Inbred Line Population

Deciphering the influence of genetics on primary metabolism in plants will provide insights useful for genetic improvement and enhance our fundamental understanding of plant growth and development. Although maize (Zea mays) is a major crop for food and feed worldwide, the genetic architecture of its primary metabolism is largely unknown. Here, we use high-density linkage mapping to dissect large-scale metabolic traits measured in three different tissues (leaf at seedling stage, leaf at reproductive stage, and kernel at 15 d after pollination [DAP]) of a maize recombinant inbred line population. We identify 297 quantitative trait loci (QTLs) with moderate (86.2% of the mapped QTL, R² = 2.4 to 15%) to major effects (13.8% of the mapped QTL, R² >15%) for 79 primary metabolites across three tissues. Pairwise epistatic interactions between these identified loci are detected for more than 25.9% metabolites explaining 6.6% of the phenotypic variance on average (ranging between 1.7 and 16.6%), which implies that epistasis may play an important role for some metabolites. Key candidate genes are highlighted and mapped to carbohydrate metabolism, the tricarboxylic acid cycle, and several important amino acid biosynthetic and catabolic pathways, with two of them being further validated using candidate gene association and expression profiling analysis. Our results reveal a metabolite-metabolite-agronomic trait network that, together with the genetic determinants of maize primary metabolism identified herein, promotes efficient utilization of metabolites in maize improvement.     

汉族人群NOS3 A-922G、NOS3 T-786C 与NOS3 G894T SNP的等位基因及其组合分布与高血压的相关性

为了分析汉族人群一氧化氮合酶基因NOS3 A-922G、NOS3 T-786C 与NOS3 G894T单核苷酸多态性（single nucleotide polymorphism,SNP）的等位基因及其组合分布与高血压病的相关性,选取无亲缘关系的高血压病人192例(男97例,女95例)以及无亲缘关系的健康个体122例（男76例,女46例）为对照组,提取静脉血白细胞基因组DNA,采用等位基因特异性引物PCR技术检测NOS3 A-922G、NOS3 T-786C 与NOS3 G894T 3个位点的基因型。其结果显示：高血压病组与对照组NOS3 G894T、NOS3A-922G及NOS3 T-786C各等位基因型及其基因单倍型频率比较无显著性差异(P>0.05)。男、女性别分层研究：无论男亚组还是女亚组均未发现NOS3 A-922G、NOS3 T-786C 与NOS3 G894T各个位点SNP与高血压病有相关性。等位基因组合分布研究发现NOS3 G894G +A-922G+T-786T组合基因型总体频率分布在高血压病组与正常对照组之间有显著性差异(P<0.05,χ2= 4.5944)。男、女性别分层研究：男亚组上述3个位点SNP的各个组合基因型分布频率在高血压病组与正常对照组之间无显著性差异(P>0.05);女亚组中携带NOS3 G894G+A-922G+T-786C 的组合基因型分布频率在高血压病组与正常对照组之间有显著性差异(P<0.01,χ2=8.502)。研究发现,在中国汉族人群中NOS3A–922 G、NOS3 T-786C 与NOS3 G894T SNP与高血压病无明确的相关性,且无性别差异。组合分布研究发现,NOS3 G894G+A-922G+T-786C 的组合基因型分布频率在高血压病女性亚组较健康女性亚组明显减低,提示携带该组合基因型女性人群可能不易患高血压病。     

Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (MutaMouse)

Quinoline is carcinogenic to the liver in rodents, but it is not clear whether it acts by a genotoxic mechanism. We previously demonstrated that quinoline does induce gene mutation in the liver of lambda/lacZ transgenic mice. In the present report, we reveal the molecular nature of the mutations induced by quinoline in the lambda cII gene, which is also a phenotypically selectable marker in the lambda transgene. (The cII gene has 294bp, which enables much easier sequence analysis than the original lacZ gene (3kb)). The liver cII mutant frequency was nine times higher in quinoline-treated mice than in control mice. Sequence analysis revealed that quinoline induced primarily G:C to C:G transversions (25 of 34). Thus, we have confirmed that quinoline is genotoxic in its target organ, and the G:C to C:G transversion is the molecular signature of quinoline-induced mutations.     

青海野生黑果枸杞再生体系的建立及遗传稳定性分析

以野生黑果枸杞(Lycium ruthenicum Murr.)的无菌苗叶片作为外植体,建立了两条再生体系:一条是经愈伤组织再分化的间接再生体系,一条是不经愈伤组织再分化的直接再生体系。并采用流式细胞术(FCM)及ISSR分子标记技术对两种途径再生苗进行了遗传稳定性分析。结果表明:(1)最佳愈伤组织诱导培养基为MS+1.5 mg·L-12,4-二氯苯氧乙酸(2,4-D),诱导率达100%;最佳分化培养基为MS+1.5 mg·L-16-苄氨基腺嘌呤(6-BA)+0.1 mg·L-1吲哚-3-丁酸(IBA),1 g愈伤组织上的平均不定芽数为39.4个。(2)叶片直接诱导不定芽的最佳培养基为MS+0.5 mg·L-16-BA+0.3 mg·L-1α-萘乙酸(NAA),不定芽诱导率为92.9%,每个外植体上平均不定芽数为18.1个。(3)两条途径再生的不定芽在不含植物生长调节剂的MS培养基上,2周内均可正常生根。(4)FCM结果显示亲本苗及2种再生苗均为二倍体。(5)ISSR分析表明,间接再生苗的平均遗传相似性系数为0.84,直接再生苗的平均遗传相似性系数为0.91,直接再生体系是一种更加快速高效的繁殖方法。