Split-seed: a new tool for maize researchers

Until recently, immature embryos have been a choice tissue for manipulation in culture for regeneration and production of transgenic maize plants. The utility of this explant has been compromised by low output, genotype dependence and time-consuming incubation in tissue culture. We have developed a new explant, the split-seed, which addresses these limitations by formally treating each seed as though it were a “dicot”. By splitting maize seed longitudinally, three different tissues: the scutellum, the coleoptilar-ring and the shoot apical meristems are simultaneously exposed. The cells of these tissues can be made competent to enhance the regeneration, given that the molecular networks resulting from exposure of the split-seed to hormones is likely to be different from whole seed and, in turn, affects the in vitro response. Using this explant, callus induction frequency exceeded 92% and the regeneration frequency was 76%. The mean number of shoots regenerated via callus was 11 shoots per callus clump and 28 shoots per explant at first sub-culture. All of the regenerated plants survived and were 95% fertile. The large numbers of fertile plants produced were regenerated in 6–8 weeks. Finally, the incidence of regenerated plants varies as a function of growth regulator profile.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Isolation of protoplasts for patch-clamp experiments: an improved method requiring minimal amounts of adult leaf or root tissue from monocotyledonous or dicotyledonous plants

Summary For the purpose of patch-clamp studies, a protoplast isolation procedure is presented in which an osmotic shock (changing the osmolarity of the medium) and centrifugation step are omitted to limit mechanical stress. Apart from the reduction of mechanical stress factors, protoplast washing is also limited. Protoplasts have been isolated from different monocotyledonous and dicotyledonous plant species and from different tissues (leaves and roots). The seal success rate in patch-clamp experiments was high (about 85% successful seals showing a seal resistance > 10G). To evaluate the electrogenic viability of the protoplasts, fusicoccin and light responses of the plasma membrane and channel activity were tested. The addition of fusicoccin to the bathing medium caused typical high activation of the proton pump. Switching the light on and off caused transient depolarizations and hyperpolarizations, respectively, matching data reported for mesophyll cells in micro-electrode studies. Whole-cell and single-channel recordings of protoplast plasma membranes isolated from intact tobacco andArabidopsis leaves were comparable with data published for protoplasts from corresponding tissue cultures. It is concluded that our isolation procedure yields protoplasts with electrogenic responses and is therefore suitable for patch-clamp studies in physiological research.Abbreviations K₄BAPTA potassium-1,2-bis(2-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid - BTP 1,3-bis[tris(hydroxy-methyl)-methylamino]propane - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid - Mes 2-(N morpholino)ethane sulphonic acid - Tris tris (hydroxymethyl)aminomethane - WC whole cell     

Identification of Horizontal Gene Transfer from Phylogenetic Gene Trees

We suggest a new procedure to search for the genes with horizontal transfer events in their evolutionary history. The search is based on analysis of topology difference between the phylogenetic trees of gene (protein) groups and the corresponding phylogenetic species trees. Numeric values are introduced to measure the discrepancy between the trees. This approach was applied to analyze 40 prokaryotic genomes classified into 132 classes of orthologs. This resulted in a list of the candidate genes for which the hypothesis of horizontal transfer in evolution looks true.     

Approaches to investigating the ecology of plasmids in marine bacterial communities

To better understand prokaryotic gene flux in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of plasmid populations in marine bacterial communities, information on the distribution, diversity, and ecological traits of marine plasmids is necessary. This mini-review highlights recent insights gained into the molecular diversity and ecology of plasmids occurring in marine microbial communities.     

Phylogeny vs genome reshuffling: horizontal gene transfer

The evolutionary events in organisms can be tracked to the transfer of genetic material. The inheritance of genetic material among closely related organisms is a slow evolutionary process. On the other hand, the movement of genes among distantly related species can account for rapid evolution. The later process has been quite evident in the appearance of antibiotic resistance genes among human and animal pathogens. Phylogenetic trees based on such genes and those involved in metabolic activities reflect the incongruencies in comparison to the 16S rDNA gene, generally used for taxonomic relationships. Such discrepancies in gene inheritance have been termed as horizontal gene transfer (HGT) events. In the post-genomic era, the explosion of known sequences through large-scale sequencing projects has unraveled the weakness of traditional 16S rDNA gene tree based evolutionary model. Various methods to scrutinize HGT events include atypical composition, abnormal sequence similarity, anomalous phylogenetic distribution, unusual phyletic patterns, etc. Since HGT generates greater genetic diversity, it is likely to increase resource use and ecosystem resilience.     

噬藻体和蓝藻间的基因转移及协同进化作用

生物物种之间的水平基因转移广泛存在于细菌、古生菌和真核生物中,并能造成同一生境中种群的快速协同进化。噬藻体是感染蓝藻的专一性病毒,近年研究表明其在蓝藻水华生消中发挥了重要作用,使人们认识到了噬藻体的重要生态地位。综述了物种间的水平基因转移,介绍了噬藻体遗传多样性研究中常用的光合作用基因、结构蛋白基因等靶标基因所介导的基因转移以及基因转移引起的病毒和宿主的协同进化,并介绍了研究基因转移所用到的试验技术以及今后所要面临的问题。     

Eukaryotic origin of a metabolic pathway in virus by horizontal gene transfer

Horizontal gene transfer, the movement of genetic materials across the normal mating barriers between organisms occurs frequently and contributes significantly to the evolution of both eukaryotic and prokaryotic genomes. However, few concurrent transfers of functionally related genes implemented in a pathway from eukaryotes to prokaryotes are observed. Here, we did phylogenetic analyses to support that the genes, i.e. dihydrofolate reductase, glycine hydroxymethyltransferase, and thymidylate synthase involved in thymidylate metabolism, in Hz-1 virus were obtained from insect genome recently by independent horizontal gene transfers. In addition, five other related genes in nucleotide metabolism show evidences of horizontal gene transfers. These genes demonstrate similar expression pattern, and they may have formatted a functionally related pathway (e.g. thymidylate synthesis, and DNA replication) in Hz-1 virus. In conclusion, we provide an example of horizontal gene transfer of functionally related genes in a pathway to prokaryote from eukaryote.     

Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae and Streptococcus oralis

Abstract The region encoding the transpeptidase domain of the penicillin-binding protein 2B (PBP 2B) gene of two penicillin-resistant clinical isolates of Streptococcus oralis was > 99.6% identical in nucleotide sequence to that of a penicillin-resistant serotype 6 isolate of Streptococcus pneumoniae . The downstream 849 base pairs of these genes were identical. Analysis of the data indicates that the PBP gene has probably been transferred from S. pneumoniae into S. oralis , rather than vice versa, and shows that one region of this resistance gene has been distributed horizontally both within S. pneumoniae and into two different viridans group streptococci.     

接合转移质粒pUCP24T的构建与性能研究

目的 构建能在大肠埃希菌(E.coli)和铜绿假单胞菌(PA)间高效接合转移的质粒.方法 通过NCBI blastn程序分析质粒pCVD442接合反应起始序列oriT,PCR扩增oriT后插入pMD18-T,再将pMD1 8-oriT转化E.coli DH5α,经酶切、测序验证后用SmaI和HindIIII双酶切亚克隆至质粒pUCP24,获得质粒pUCP24T,将pUCP24T转化E.coli后研究pUCP24T从E.coli到PA的接合效率和质粒稳定性等.结果 成功构建pUCP24T重组质粒,在E.coli和PA的接合实验中,E.coli(pUCP24T)-PA共培养2h的接合效率平均为3.588×10-2,按12 h一代连续9代继代培养108 h,抗生素压力下质粒保存率为98.29％,无抗生素的培养基中质粒保存率为21.76％.结论 成功构建高接合效率的接合转移质粒pUCP24T.     

Reservoirs of Antibiotic Resistance Genes

A potential concern about the use of antibiotics in animal husbundary is that, as antibiotic resistant bacteria move from the farm into the human diet, they may pass antibiotic resistance genes to bacteria that normally reside in a the human intestinal tract and from there to bacteria that cause human disease (reservoir hypothesis). In this article various approaches to evaluating the risk of agricultural use of antibiotics are assessed critically. In addition, the potential benefits of applying new technology and using new insights from the field of microbial ecology are explained.     

Presence of streptococcal pyrogenic exotoxin A and C genes in human isolates of group G streptococci

The bacteriophage-associated genes speA and speC encode streptococcal pyrogenic exotoxins of group A streptococci (GAS). Human isolates of group C and G streptococci (GCS and GGS) are commensals and the closest known genetic relatives of GAS; on occasion, GCS-GGS can cause infection that is clinically similar to GAS disease. Thirty-four human isolates of GCS-GGS were tested for speA and speC. Two GGS isolates harbored speA only, whereas a third GGS had both genes. All spe alleles found in GGS were identical to known spe alleles of GAS, except for one speA allele, which was unique. The presence of shared speA and speC alleles in GAS and GGS is highly suggestive of recent interspecies transfer. Acquisition of GAS-like virulence genes by GGS may lead to enhanced pathogenicity in this usually commensal-like organism.     

Diverse symbiovars nodulating cowpea (Vigna unguiculata L. Walp.) in highly adaptable agro-ecological zones in Mozambique

The presence of effective microsymbionts in the soil and their compatibility with the host plant are the key determinants to the N₂ fixation process. In Sub-Saharan Africa, nitrogen fixation in locally adapted cowpea and the distribution of their symbiovars are not well understood. The Aim of the study was to assess the distribution and symbiotic phylogenetic position of cowpea microsymbionts. Root nodules were sampled from various cowpea genotypes planted in Agro-Ecological Zone 7 and 8 (AEZ 7 and AEZ 8). Root-nodule bacteria were isolated and their molecular characterization was conducted. Physicochemical properties of soil were recorded. Enterobacterial Repetitive Intergenic Consensus (ERIC) distribution patterns in rhizobial genomes resulted in genetically diverse rhizobial population in Northern Mozambique. Principal component analysis showed that location-specific soil environment determined the presence of particular microsymbionts. Based on 16S rRNA and symbiotic gene analysis many diverse symbiovars were found in Mozambican soils. With few discrepancies, the results further confirmed the coevolution of the nifH, nodD, nodC and nodY/K genes, which was indicative of natural events such as vertical/horizontal gene transfer. The results suggested that ecological and phylogenetic studies of the microsymbionts are necessary to better reflect symbiovar identification and the ecological adaptation of the cowpea-nodulating rhizobial community.     

A likelihood framework to measure horizontal gene transfer

We suggest a likelihood-based approach to estimate an overall rate of horizontal gene transfer (HGT) in a simplified setting. To this end, we assume that the number of occurring HGT events within a given time interval follows a Poisson process. To obtain estimates for the rate of HGT, we simulate the distribution of tree topologies for different numbers of HGT events on a clocklike species tree. Using these simulated distributions, we estimate an HGT rate for a collection of gene trees representing a set of taxa. As an illustrative example, we use the "Clusters of Orthologous Groups of proteins" (COGs). We also perform a correction of the estimated rate taking into account the inaccuracies due to gene tree reconstructions. The results suggest a corrected HGT rate of about 0.36 per gene and unit time, in other words 11 HGT events have occurred on average among the 44 taxa of the COG species tree. A software package to estimate an HGT rate is available online (http://www.cibiv.at/software/hgt/).     

基因转移因子———一类在海洋中广泛存在的介导水平基因转移的新型生物因子

基因转移因子(Gene Transfer Agent,GTA)是一种由细菌释放的、形态和有尾病毒类似的生物颗粒。GTA颗粒携带的遗传物质是宿主基因组的随机小片段而不包含编码GTA自身的基因或病毒基因组。根据4个模式菌株释放的GTA的研究,GTA具有高效的,种间介导基因水平转移的功能。近年来大规模细菌基因组测序,发现编码GTA的基因簇广泛存在于海洋细菌基因组上,GTA是在海洋环境中发生水平基因转移的重要模式。本文在总结4个模式菌株释放的GTA的认识的基础上,着重描述海洋主要类群的细菌释放的GTA的特征,讨论在海洋生态系统中,GTA对水平基因转移的贡献,并对未来的研究进行了展望。