Possible Host Adaptation as an Evolution Factor of Cowpea aphid‐borne mosaic virus Deduced by Coat Protein Gene Analysis

Cowpea aphid‐borne mosaic virus (CABMV) causes major diseases in cowpea and passion flower plants in Brazil and also in other countries. CABMV has also been isolated from leguminous species including, Cassia hoffmannseggii, Canavalia rosea, Crotalaria juncea and Arachis hypogaea in Brazil. The virus seems to be adapted to two distinct families, the Passifloraceae and Fabaceae. Aiming to identify CABMV and elucidate a possible host adaptation of this virus species, isolates from cowpea, passion flower and C. hoffmannseggii collected in the states of Pernambuco and Rio Grande do Norte were analysed by sequencing the complete coat protein genes. A phylogenetic tree was constructed based on the obtained sequences and those available in public databases. Major Brazilian isolates from passion flower, independently of the geographical distances among them, were grouped in three different clusters. The possible host adaptation was also observed in fabaceous‐infecting CABMV Brazilian isolates. These host adaptations possibly occurred independently within Brazil, so all these clusters belong to a bigger Brazilian cluster. Nevertheless, African passion flower or cowpea‐infecting isolates formed totally different clusters. These results showed that host adaptation could be one factor for CABMV evolution, although geographical isolation is a stronger factor.     

Rapid detection of cowpea aphid-borne mosaic virus in cowpea seeds

Four cultivars of cowpea (Vigna unguiculata [L]. Walp.) were infected with cowpea aphid-borne mosaic virus (CABMV) by natural infection in field plots. Seeds taken from these plants were tested for the presence of the virus by ELISA and symptom observation on the plantlets grown from the seeds. A biotin/ streptavidin ELISA technique was used and found to be more sensitive than a standard ELISA protocol for detecting CABMV infection in seed. There was a good correlation between the ELISA detection of CABMV in tissue taken from single cowpea seeds and subsequent development of infected plants grown from the same seeds. The ELISA technique is reliable for selecting CABMV-free stocks of cowpea seeds.     

Alarming increase in the incidence of Cucumber mosaic virus in cowpea (Vigna unguiculata (L.) Walp.) in northern Nigeria

Cowpea plays a key nutritional role in the diet of the Nigerian people. Viral diseases are a major limitation to cowpea production worldwide, and thus, constant viral surveillance is crucial for monitoring and management purposes. In this study, cowpea leaf samples from fields in three northern Nigeria states, Kano, Kaduna and Niger, were tested to determine the status of six common viruses previously reported in these cowpea-producing states following the release of virus-resistant varieties. Cowpea aphid-borne mosaic virus (CABMV), Blackeye cowpea mosaic virus (BICMV), Cowpea mottle virus, Southern bean mosaic virus and Cucumber mosaic virus (CMV) were detected. Cowpea yellow mosaic virus, which was previously reported in all three states, was not detected in any of the samples tested, while CMV that was previously regarded as unimportant to cowpea production in Nigeria had the highest incidence in all three states, and the overall highest incidence of 58.8%, while CABMV had the lowest incidence (7.5%). CMV was also present in seven of the ten mixed infection combinations detected. Dual infection of CMV and BICMV, which often results in cowpea stunt, the most devastating cowpea disease in the USA, was the most frequently detected mixed infection (28.1%) and was detected in all three states. This observed elevation in CMV infection in cowpea must be closely monitored and swiftly managed to avert possible devastating crop yield losses.     

Evaluation of hybridization conditions for spotted oligonucleotide-based DNA microarrays

We compared different hybridization conditions of oligonucleotide-based DNA microarray to acquire optimized and reliable microarray data. Several parameters were evaluated at different hybridization conditions, including signal-to-background (S:B) ratios, signal dynamic range, usable spots, and reproducibility. Statistical analysis showed that better results were obtained when spotted, presynthesized long oligonucleotide arrays were blocked with succinic anhydride and hybridized at 42°C in the presence of 50% formamide.     

基因芯片技术在检测肠道致病菌方面的应用

基因芯片技术具有高通量、自动化、快速检测等特点,因此被广泛地应用于各种研究领域,如细菌分子流行病学、细菌基因鉴定、致病分子机理、基因突变及多态性分析、表达谱分析、DNA测序和药物筛选等。现介绍基因芯片检测肠道致病菌方面的国外研究进展,基因芯片应用于检测肠道致病菌的3个方面:结合多重PCR对致病菌的毒力因子或者特异性基因进行鉴定;直接检测细菌的DNA或者RNA;以致病细菌核糖体RNA作为检测的靶基因同时检测多种肠道致病菌。由于其检测的高效率,该技术要优于其他分子生物学检测方法。基因芯片技术在肠道致病菌检测中有着巨大的应用价值,具有广阔的应用前景。     

DNA methylation inhibits propagation of tomato golden mosaic virus DNA in transfected protoplasts

The effects of methylation on plant viral DNA replication have been studied inNicotiana tabacum protoplasts transfected with DNA of the geminivirus tomato golden mosaic virus (TGMV). The transfected cells were also used to determine whether experimentally introduced methylation patterns are maintained in extrachromosomal viral DNA. Replacement of cytosine residues with 5-methylcytosine (m⁵C) reduced the amount of viral DNA which accumulated in transfected protoplasts. The reduction was observed whether m⁵C residues were substituted for cytosine residuesin vitro in either the viral strand or the complementary strand of double-stranded circular inoculum DNAs containing tandemly repeated copies of the A component of the TGMV genome. Both limited and extensive cytosine methylation of TGMV DNA sequencesin vitro was not propagated in progeny viral DNA. The absence of detectable maintenance-type methylation of the transfecting TGMV DNA sequences may be related to the lack of methylation observed in double-stranded TGMV DNA isolated from infected plants.     

Replicative form DNA of abutilon mosaic virus is present in plastids

Summary Abutilon mosaic virus (AbMV), a geminivirus, is so far the only plant virus for which transfer of its single-stranded (ss) DNA to plastids has been proven. We now show that both parts of the genome (DNA-1 and DNA-2) are present in these organelles as well as the double-stranded replicative form DNA (RF-DNA) intermediates. Artefactual copurification of the RF-DNA with the plastids was excluded by DNase I studies. The appearance of RF-DNA in plastids was found to be light dependent.     

Cauliflower mosaic virus as a gene expression vector for plants

It is possible to replace the CaMV (cauliflower mosaic virus) ORF (open reading frame) II with foreign sequences without interfering with virus viability. Such recom-binants can induce the synthesis of substantial amounts of a foreign protein in infected plants and confer new properties to these plants. However, so far only three genes have been successfully cloned and expressed in this way. The expression mechanism of CaMV demands precise replacement of ORF II and probably certain structural features of the viral 35S RNA, which should not be disturbed by inserted sequences. Since these features are largely unknown, it cannot at present be pre-dicted whether an insert will be tolerated. It is more likely that larger inserts will disturb the viral gene expression mechanism than smaller ones.     

Coating of nitrocellulose for colorimetric DNA microarrays

We report on the modification of a nitrocellulose film with copoly(DMA-NAS-MAPS), a tercopolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS). The chains of this polymer, interacting with nitrocellulose fibers, introduce active ester functionalities that promote the covalent binding of short oligonucleotide fragments to the nitrocellulose thin film. Using colorimetric detection, naked eye visible DNA microarrays are developed for easy identification of foodborne pathogens. The fast and robust procedure of nitrocellulose functionalization opens the opportunity to implement this material in disposable analytical microdevices that do not require sophisticated readout systems.     

Cauliflower mosaic virus 35 S promoter directs S phase specific expression in plant cells

Summary An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.     

Novel developments for improved detection of specific mRNAs by DNA chips

Microarrays have revolutionized gene expression analysis as they allow for highly parallel monitoring of mRNA levels of thousands of genes in a single experiment. Since their introduction some 15 years ago, substantial progress has been achieved with regard to, e.g., faster or more sensitive analyses. In this review, interesting new approaches for a more sensitive detection of specific mRNAs will be highlighted. Particularly, the potential of electrical DNA chip formats that allow for faster mRNA analyses will be discussed.     

甘蔗花叶病毒CP基因的高效表达和抗血清制备

将甘蔗花叶病毒的CP基因克隆到表达载体pET22b( ),并在大肠杆菌BL21(DE3)中得到高效表达,表达蛋白约占总蛋白的15％。用此蛋白制备了效价高专化性强的抗体。此方法可以解决制备马铃薯Y病毒属病毒的血清时遇到的病毒提纯产量低和寄主蛋白影响等问题。     

Effect of deletions in the cauliflower mosaic virus polyadenylation sequence on the choice of the polyadenylation sites in tobacco protoplasts

Summary Deletions were made in the cauliflower mosaic virus polyadenylation sequence which was cloned downstream of the -glucuronidase gene (gus). The populations of mRNAs generated in tobacco mesophyll protoplasts by transient expression with the various constructs were analysed using a polymerase chain reaction procedure. When no deletion was present in the sequence, the mRNA appeared to be polyadenylated at two major polyadenylation sites. A deletion upstream from the AATAAA sequence made the population of polyadenylated mRNAs very heterogenous at their 3 ends. A deletion downstream of the AATAAA sequence had no effect on the choice of the site. Alternative polyadenylation sites were used when the native polyadenylation site was deleted. These results are discussed in relation to data obtained with other polyadenylation sequences from both plants and animals.     

Physical map of DNA from a new cauliflower mosaic virus strain

The restriction enzymes AluI, BamHI, BglII, EcoRI, HindIII, and SalI have been used to characterize and map a new cauliflower mosaic virus strain (Cabb-S). These fragments have been ordered by examining their overlapping regions after double enzymatic digestion. The single SalI cleavage site was chosen as the point of origin. We compare this strain with those already described.     

Expression of tobacco mosaic virus RNA in transgenic plants

Summary Tobacco mosaic virus (TMV) is a message-sense, single-stranded RNA virus that infects many Solanaceae plants. A full-length cDNA copy of TMV genomic RNA was constructed and introduced into the genomic DNA of tobacco plants using a disarmed Ti plasmid vector. Transformed plants showed typical symptoms of TMV infection, and their leaves contained infectious TMV particles. This is the first example of the expression of RNA virus genomic RNAs in planta.     

Adaptable gene‐specific dye bias correction for two‐channel DNA microarrays

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA‐bound proteins. DNA microarrays can suffer from gene‐specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene‐ And Slide‐Specific Correction, GASSCO) is presented, whereby sequence‐specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence‐based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available.     

Respiratory syncytial virus infection activates STAT signaling in human epithelial cells

Acute respiratory syncytial virus (RSV) infection causes airway inflammation and exacerbates asthma, but the mechanism of inflammation is poorly understood. The role of the STAT-signaling pathway in RSV infection in epithelial cells was examined in this study. DNA microarray analyses of RSV-infected human alveolar type II (A549) epithelial cells identified several genes whose expression was altered from -5.5 to +56.4-fold. Four of the highly expressed genes contained STAT-binding elements. In A549 and normal human bronchial epithelial cells (NHBE), RSV induced phosphorylation and nuclear translocation of STAT-1alpha that was abrogated when RSV attachment was blocked. Treatment with a JAK-2 inhibitor or transfection with dominant-negative STAT-1alpha blocked STAT-1alpha activation and RSV infection. RSV also activated STAT-3 and IL-6 specific antibodies blocked this activation. Thus, activation of the STAT-1alpha and STAT-3 pathways play a role in RSV infection.     

Development of a spot reliability evaluation score for DNA microarrays

We developed a reliability index named SRED (Spot Reliability Evaluation Score for DNA microarrays) that represents the probability that the calibrated gene expression level from a DNA microarray would be less than a factor of 2 different from that of quantitative real-time polymerase chain reaction assays whose dynamic quantification range is treated statistically to be similar to that of the DNA microarray. To define the SRED score, two parameters, the reproducibility of measurement value and the relative expression value were selected from nine candidate parameters. The SRED score supplies the probability that the expression level in each spot of a microarray is less than a certain-fold different compared to other expression profiling data, such as QRT-PCR. This score was applied to 1,500,000 points of the expression profile in the RIKEN Expression Array Database.