Gene replacement by homologous recombination in the multicellular green alga Volvox carteri

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Inhibition of apoptosis by overexpressing Bcl-2 enhances gene amplification by a mechanism independent of aphidicolin pretreatment.

To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.

Multiple analysis of three common genetic alterations associated with thrombophilia

We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia. (C) 2000 Lippincott Williams and Wilkins.

No association between MTHFR A1298C and MTRR A66G polymorphisms, and MS in an Australian cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ2 = 0.005, P = 0.95, MTHFR χ2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS

Effects of dietary folate intake on migraine disability and frequency

Objective Migraine is a highly disabling disease affecting a significant proportion of the Australian population. The Methylenetetrahydrofolate Reductase (MTHFR) C677T variant has been associated with increased levels of homocysteine and risk of migraine with aura (MA). Folic acid, Vitamin B6 and B12 supplementation has been previously shown to reduce increased levels of homocysteine and decrease migraine symptoms. However the influence of dietary folate intake on migraine has been unclear. The aim of the current study was to analyse the association of dietary folate intake in the form of dietary folate equivalent (DFE), folic acid (FA) and total food folate (TFF) on migraine frequency, severity and disability. Methods A cohort of 141 adult females of Caucasian descent with MA was genotyped for the MTHFRC677T variant using restriction enzyme digestion. Dietary folate information was collected from all participants and analysed using the “FoodWorks” 2009 package. Folate consumption was compared to migraine frequency, severity and disability using linear regression. Results A significant inverse relation was observed between DFE [R2= 0.201, P= 0.045, CI (-0.004, -0.001)] and FA [R2= 0.255, P= 0.036, 95% CI (-0.009, -0.002)] consumption and migraine frequency. It was also observed that in individuals with the CC genotype for the MTHFR C677T variant, migraine frequency was significantly linked to FA consumption [R2= 0.077, P= 0.029, CI (-0.009, -0.005)]. Conclusions The results from this study indicate that folate intake in the form of folic acid may influence migraine frequency in female MA sufferers.

A transcriptome resource for the koala (Phascolarctos cinereus) : insights into koala retrovirus transcription and sequence diversity

Background The koala, Phascolarctos cinereus, is a biologically unique and evolutionarily distinct Australian arboreal marsupial. The goal of this study was to sequence the transcriptome from several tissues of two geographically separate koalas, and to create the first comprehensive catalog of annotated transcripts for this species, enabling detailed analysis of the unique attributes of this threatened native marsupial, including infection by the koala retrovirus. Results RNA-Seq data was generated from a range of tissues from one male and one female koala and assembled de novo into transcripts using Velvet-Oases. Transcript abundance in each tissue was estimated. Transcripts were searched for likely protein-coding regions and a non-redundant set of 117,563 putative protein sequences was produced. In similarity searches there were 84,907 (72%) sequences that aligned to at least one sequence in the NCBI nr protein database. The best alignments were to sequences from other marsupials. After applying a reciprocal best hit requirement of koala sequences to those from tammar wallaby, Tasmanian devil and the gray short-tailed opossum, we estimate that our transcriptome dataset represents approximately 15,000 koala genes. The marsupial alignment information was used to look for potential gene duplications and we report evidence for copy number expansion of the alpha amylase gene, and of an aldehyde reductase gene. Koala retrovirus (KoRV) transcripts were detected in the transcriptomes. These were analysed in detail and the structure of the spliced envelope gene transcript was determined. There was appreciable sequence diversity within KoRV, with 233 sites in the KoRV genome showing small insertions/deletions or single nucleotide polymorphisms. Both koalas had sequences from the KoRV-A subtype, but the male koala transcriptome has, in addition, sequences more closely related to the KoRV-B subtype. This is the first report of a KoRV-B-like sequence in a wild population. Conclusions This transcriptomic dataset is a useful resource for molecular genetic studies of the koala, for evolutionary genetic studies of marsupials, for validation and annotation of the koala genome sequence, and for investigation of koala retrovirus. Annotated transcripts can be browsed and queried at http://koalagenome.org

Signaling pathway genes for blood pressure, folate and cholesterol levels among hypertensives : an epistasis analysis

Irregular atrial pressure, defective folate and cholesterol metabolism contribute to the pathogenesis of hypertension. However, little is known about the combined roles of the methylenetetrahydrofolate reductase (MTHFR), apolipoprotein-E (ApoE) and angiotensin-converting enzyme (ACE) genes, which are involved in metabolism and homeostasis. The objective of this study is to investigate the association of the MTHFR 677 C>T and 1298A>C, ACE insertion–deletion (I/D) and ApoE genetic polymorphisms with hypertension and to further explore the epistasis interactions that are involved in these mechanisms. A total of 594 subjects, including 348 normotensive and 246 hypertensive ischemic stroke subjects were recruited. The MTHFR 677 C>T and 1298A>C, ACE I/D and ApoEpolymorphisms were genotyped and the epistasis interaction were analyzed. The MTHFR 677 C>T and ApoE polymorphisms demonstrated significant associations with susceptibility to hypertension in multiple logistic regression models, multifactor dimensionality reduction and a classification and regression tree. In addition, the logistic regression model demonstrated that significant interactions between the ApoE E3E3, E2E4, E2E2 and MTHFR 677 C>T polymorphisms existed. In conclusion, the results of this epistasis study indicated significant association between the ApoE and MTHFR polymorphisms and hypertension.

近零磁场对拟南芥生长发育影响初探及趋磁细菌(Magnetospirillum magneticum）铁还原酶基因 MmFre 的克隆和功能分析

地磁场伴随着生命的起源、发生和进化，地球上的一切生命无时无刻不处于地球磁场中。地球自诞生以来，地磁场的强度一直在细微变化，但地磁场强度减弱会对植物产生什么样的直接影响尚知之甚少。随着对太空探索的不断发展，人类越来越需要了解处于零磁场环境的太空中生物的适应性。近零磁场是地磁场的恒定组分降低为零或者接近零的空间。本论文利用近零磁场环境探索了地磁场减弱对拟南芥整个生长周期的影响，开展了近零磁场下拟南芥短期生长试验，主要包括种子的萌发、暗培养、根生长、幼苗鲜重和根向重性分析，以及对近零磁场下拟南芥整个生长周期的表型进行了观察统计分析。结果发现(1)无论在光照还是暗培养的环境中，近零磁场对拟南芥种子的萌发、幼苗根的伸长、鲜重变化以及根向重性等的影响较小。(2)对拟南芥整个生长周期过程中表型变化进行的观察和统计分析发现：近零磁场环境中，拟南芥可以完成正常的生活史;但植株开花时间推迟、开花持续时间延长、枝条数减少、植株高度受到了抑制，种子千粒重降低。表明近零磁场对拟南芥营养生长的影响较小，而对生殖生长的影响较明显，暗示地磁场作为环境因子可能参与影响植物的生殖生长。 趋磁细菌(Magnetospirillum magneticum)是一种可以沿磁力线方向运动的特殊的细菌，其胞内铁含量是菌体干重的3％，是非磁性细菌的数百倍，其中的铁主要以Fe3O4/Fe3S4 形式存在于磁小体(magnetosome) 中。趋磁细菌主要通过分泌转铁载体吸收环境中的三价铁。在磁小体合成过程中，三价铁还原为二价是一个必需的过程，因而铁还原酶在趋磁细菌的铁还原过程中可能起着重要的作用。我们以趋磁细菌AMB-1 为材料，克隆了预测的铁还原酶基因，命名为MmFre ，并在内源铁还原酶活性较低的酵母突变株S288C fre1 fre2 中进行异源超表达，对其铁还原活性进行了初步分析;同时结合GFP 融合蛋白技术对该基因的表达产物进行了酵母的亚细胞定位。结果表明：(1)利用生物信息学分析发现，MmFre 基因编码区含有1335 bp，编码444 个氨基酸残基;氨基酸序列中含有一个FAD 结合位点，并具有6 个跨膜结构域;(2)该基因在酵母表达后利用酵母活体进行酶活性检测发现，其铁还原酶活性是对照组的4 倍，暗示该基因在真核生物中的表达产物可以执行铁还原的功能;(3)利用激光共聚焦显微镜观察发现该基因的表达产物与GFP 构成的融合蛋白广泛的定位在细胞的膜上。因而，MmFre 基因的表达产物确实具有铁还原酶活性，且没有膜特异性分布，其对趋磁细菌磁小体生物合成中铁的还原可能起着重要作用。

拟南芥砷酸还原酶基因AtACR2的表达调控及其功能分析

将砷酸盐还原为亚砷酸盐是植物砷代谢途径中的关键步骤，其中砷酸还原酶是催化砷酸盐还原的关键酶。目前，对植物中砷酸还原酶基因的表达调控机制及该基因的功能了解得还不是很清楚。因此研究拟南芥砷酸还原酶基因的表达调控及其功能对于探讨植物对砷吸收、代谢、转运和富集的分子机制有重要意义。 本论文利用拟南芥砷酸还原酶基因（AtACR2）的启动表达调控序列的不同组合驱动GUS基因转录表达，对AtACR2启动表达调控序列的功能进行了分析；同时利用过表达、AtACR2基因T-DNA插入缺失突变体和地上部特异表达对拟南芥和蜈蚣草砷酸还原酶的基因功能进行了初步分析，主要结果如下： 1．对拟南芥AtACR2基因在不同砷酸盐处理浓度(0、100 yM、200 yM) 下的RT-PCR分析初步表明：在未用Na3As04处理的拟南芥幼苗中，AtACR2基因在根和茎叶中均有表达，且其在根中的转录水平高于茎叶中。同时该基因的表达在转录水平上受砷酸盐的负调控，即随着外界砷酸盐浓度的升高，AtACR2基因的转录水平降低。 2．将AtACR2基因不同启动调控序列组合驱动GUS基因转录表达，结果表 明：①由AtACR2基因上游1250 bp及其5’端非编码医构成的启动调控序列不足以启动AtACR2基因的转录表达和砷酸盐胁迫的应答；②在第一外显子和第一内含子中存在启动AtACR2基因起始转录表达的关键序列元件，它们的存在决定了该基因能否得以转录表达；⑧第一外显子和第一内含子序列中不仅存在起始基因转录的必需元件，还存在砷胁迫相关的应答元件，参与砷酸盐抑制AtACR2基因的转录表达调控；④在第二外显子和第二内含子中可能存在增强基因表达的调控元件序列，进一步影响该基因转录表达强度的调控。 3． 拟南芥AtA CR2基因和砷超富集植物蜈蚣草PvA CR2基因在拟南芥中过表达后的功能分析初步表明：①转基因植株能够通过减少体内As含量增强对砷酸盐的抗性；②两种植物的砷酸还原酶作用能力存在一定差异，其中超表达蜈蚣草PvA CR2能够使转基因植株根中As含量更少，但其对砷酸盐胁迫的抗性并没有AtACR2超表达植株强，这可能与转 PvA CR2基因植株地上部积累相对较高的砷含量有关。 4．将AtA CR2和PvA CR2在拟南芥中地上部特异表达后，抗性实验初步表明：①以野生型拟南芥为背景材料进行地上部特异超表达AtACR2或PvA CR2基因，不能增强转基因植株对砷酸盐抗性；②以AtA CR2基因的T-DNA插入缺失突变体为背景材料地上部特异表达AtACR2或PvA CR2基因，却能够明显增强转基因植株对砷酸盐的抗性。综上所述，植物砷酸还原酶基因在植物对砷酸盐胁迫的响应和调控中起着重要作用。

A population model of folate-mediated one-carbon metabolism.

BACKGROUND: Previous mathematical models for hepatic and tissue one-carbon metabolism have been combined and extended to include a blood plasma compartment. We use this model to study how the concentrations of metabolites that can be measured in the plasma are related to their respective intracellular concentrations. METHODS: The model consists of a set of ordinary differential equations, one for each metabolite in each compartment, and kinetic equations for metabolism and for transport between compartments. The model was validated by comparison to a variety of experimental data such as the methionine load test and variation in folate intake. We further extended this model by introducing random and systematic variation in enzyme activity. OUTCOMES AND CONCLUSIONS: A database of 10,000 virtual individuals was generated, each with a quantitatively different one-carbon metabolism. Our population has distributions of folate and homocysteine in the plasma and tissues that are similar to those found in the NHANES data. The model reproduces many other sets of clinical data. We show that tissue and plasma folate is highly correlated, but liver and plasma folate much less so. Oxidative stress increases the plasma S-adenosylmethionine/S-adenosylhomocysteine (SAM/SAH) ratio. We show that many relationships among variables are nonlinear and in many cases we provide explanations. Sampling of subpopulations produces dramatically different apparent associations among variables. The model can be used to simulate populations with polymorphisms in genes for folate metabolism and variations in dietary input.

Moderately elevated plasma homocysteine, MTHFR genotype and risk for stroke, vascular dementia and alzheimer's disease in northern ireland

BACKGROUND AND PURPOSE: Elevated plasma homocysteine level has been associated with increased risk for cardiovascular and cerebrovascular disease. Variation in the levels of this amino acid has been shown to be due to nutritional status and methylenetetrahydrofolate reductase (MTHFR) genotype. METHODS: Under a case-control design we compared fasting levels of homocysteine and MTHFR genotypes in groups of subjects consisting of stroke, vascular dementia (VaD), and Alzheimer disease patients and normal controls from Northern Ireland. RESULTS: A significant increase in plasma homocysteine was observed in all 3 disease groups compared with controls. This remained significant after allowance for confounding factors (age, sex, hypertension, cholesterol, smoking, creatinine, and nutritional measures). MTHFR genotype was not found to influence homocysteine levels, although the T allele was found to increase risk for VaD and perhaps dementia after stroke. CONCLUSIONS: We report that moderately high plasma levels of homocysteine are associated with stroke, VaD, and Alzheimer disease. This is not due to vascular risk factors, nutritional status, or MTHFR genotype

BELFAST centenarians: A Case of Optimal Cardiovascular Risk?

Introduction: Centenarians are reservoirs of genetic and environmental information to successful ageing and local centenarian groups may help us to understand some of the factors that contribute to longevity. The current centenarian cohort in Belfast survived the 1970s epidemic of death from coronary heart disease in Northern Ireland, where cardiovascular mortality was almost highest in the world. These centenarians provided an opportunity to assess biological and genetic factors important in cardiovascular risk and ageing. Methods: Thirty-five (27 female, 8 male) centenarians, participants of the Belfast Elderly Longitudinal Free-living Ageing STudy (BELFAST), were community-living and of good cognition at enrolment. Results: Centenarians showed median Body Mass Index (BMI) at 25.7, systolic blood pressure 140mmHg and diastolic blood pressure 90mmHg, and fasting glucose of 5.54 mmol/l with no sex-related difference. Lipoproteins showed median cholesterol 5.3, High Density Lipoprotein (HDL) 1.10 and Low Density Lipoprotein (LDL) 3.47umol/l respectively. Centenarian smokers showed no different blood pressure or lipid measurements compared with non-smokers. Malondialdehyde, a measure of lipid peroxidation, was low at 1.19 umol/l, and measures of antioxidant status were varied. Male centenarians did not carry any of the vascular risk genotypes studied-ApoE4 for Apolipoprotein E (ApoE), DD for Angiotensinogen Converting Enzyme (ACE) and tt for 5,10-methylenetetrahydrofolate reductase (MTFHR), though this was not true for female centenarians.. Conclusions: This small local study shows that Belfast centenarians carry a reasonably favourable risk profile, except for age, with respect to cardiovascular disease. There is also some evidence that vascular risk factors and genotypes may be tolerated differently between the male and female centenarians. Maintaining a favourable cardiovascular risk profile seems likely to improve the chance of becoming a centenarian, especially for males.

Influences of phosphorus starvation on OsACR2.1 expression and arsenic metabolism in rice seedlings

The effects of phosphorus (P) status on arsenate reductase gene (OsACR2.1) expression, arsenate reductase activity, hydrogen peroxide (H(2)O(2)) content, and arsenic (As) species in rice seedlings which were exposed to arsenate after -P or +P pretreatments were investigated in a series of hydroponic experiments. OsACR2.1 expression increased significantly with decreasing internal P concentrations; more than 2-fold and 10-fold increases were found after P starvation for 30 h and 14 days, respectively. OsACR2.1 expression exhibited a significant positive correlation with internal root H(2)O(2) accumulation, which increased upon P starvation or exposure to H(2)O(2) without P starvation. Characterization of internal and effluxed As species showed the predominant form of As was arsenate in P-starved rice root, which contrasted with the +P pretreated plants. Additionally, more As was effluxed from P-starved rice roots than from non-starved roots. In summary, an interesting relationship was observed between P-starvation induced H(2)O(2) and OsACR2.1 gene expression. However, the up-regulation of OsACR2.1 did not increase arsenate reduction in P-starved rice seedlings when exposed to arsenate.

Renal failure after high-dose methotrexate in a child homozygous for MTHFR C677T polymorphism

We report the case of an 11-year-old female treated for mediastinal T-cell lymphoma who presented renal failure following the second cycle of high-dose methotrexate (HDMTX). Because of life threatening plasma methotrexate (MTX) levels, carboxypeptidase G2 (CPDG2) was administered resulting in a dramatic decrease within 1 hr. The patient recovered from renal failure and no other side effects were observed. Homozygosity for the methylentetrahydrofolate reductase (MTHFR) C677T polymorphism diagnosed by molecular genetic analysis was the only explanation for this toxicity.