Towards Efficient Isolation of R Gene Orthologs from Multiple Genotypes: Optimization of Long Range-PCR

Resistance (R) genes and the proteins they encode are key components of the defense system of plants. The exploration of R gene diversity enables the study of R gene evolution and may facilitate the isolation of new and functional alleles. Most cloned R genes occur in clusters of related sequences. Thus, the development of a tool for reliable recovery of orthologous R gene sequences to the exclusion of paralogous sequences will facilitate R gene diversity analysis. The late blight resistance gene RB is a single functional locus embedded within a cluster of related sequences. Previously, the functional RB allele was cloned from wild potato using a Long Range-PCR (LR-PCR) technique, suggesting this method may be a promising tool for recovery of R gene orthologs in other genotypes. Using the RB gene as a model, we explored the limitations and improved three technical aspects of LR-PCR for multi-genotype applications. We present improved primers for the recovery of the RB locus and have identified efficient DNA extraction procedures and reliable amplification systems. We document that consensus sequences built from three independently generated LR-PCR clones can be up to 100% accurate. Our results show encouraging advances toward successful application of LR-PCR for isolating alleles from orthologous R gene loci.     

Effects of gap size and associated changes in light and soil moisture on the understorey vegetation of a Hungarian beech forest

In European beech forests windstorms often create canopy gaps and change the level of incident light, soil moisture and nutrient availability on the forest floor. Understanding the interrelations between gap size and environmental change, and the effects these have on regeneration processes is a prerequisite for developing techniques of nature-based forestry. The aims of this study were to investigate the effects of gap size on the resulting spatial distributions of key abiotic environmental variables (light and soil moisture) in gaps, and to study how light and soil moisture affect the abundance and distribution of herb layer species. To do this we used eight artificially created gaps – three large (diameter: 35–40 m) and five small (diameter: 10–15 m) – in a mesotrophic submontane beech forest. Data on species’ importance and substrate types were collected in systematically distributed 1 m×1 m quadrats before gap creation and on four occasions during the next two growing seasons. Hemispherical photographs were taken and analysed to estimate relative light intensity. Soil moisture was measured by frequency domain and capacitance probes. It was found that gap size had a profound effect on the environmental variables measured. While relative light intensity values in small gaps did not reach those in large gaps, soil moisture levels did reach similar maximum values in gap centres regardless of gap size. Richness, composition and total cover of herbaceous vegetation were different in small versus large gaps. Much of this difference was attributed to the presence of specific relative light intensities and also to the increased amount of available soil moisture in gaps. Species were differently affected by the combined effects of light and soil moisture, as well as by differences in available substrates. All this resulted in species-specific distribution patterns within gaps.     

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C₄ NADP-malic enzyme species in which the C₄ and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C₄ system. Using differential display, mRNA populations from C₃ and C₄ H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C₄ leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C₄ upregulated genes were identified. Among these one encoded a previously recognized C₄ phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C₄ system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C₄ system in a C₃ crop species.Srinath K. Rao and Hiroshi Fukayama contributed equally to this study.     

Interactions of earthworms with Atrazine-degrading bacteria in an agricultural soil

In the last 10 years, accelerated mineralization of Atrazine (2-chloro-ethylamino-6-isopropylamino-s-triazine) has been evidenced in agricultural soils repeatedly treated with this herbicide. Here, we report on the interaction between earthworms, considered as soil engineers, and the Atrazine-degrading community. The impact of earthworm macrofauna on Atrazine mineralization was assessed in representative soil microsites of earthworm activities (gut contents, casts, burrow linings). Soil with or without earthworms, namely the anecic species Lumbricus terrestris and the endogenic species Aporrectodea caliginosa, was either inoculated or not inoculated with Pseudomonas sp. ADP, an Atrazine-degrading strain, and was either treated or not treated with Atrazine. The structure of the bacterial community, the Atrazine-degrading activity and the abundance of atzA, B and C sequences in soil microsites were investigated. Atrazine mineralization was found to be reduced in representative soil microsites of earthworm activities. Earthworms significantly affected the structure of soil bacterial communities. They also reduced the size of the inoculated population of Pseudomonas sp. ADP, thereby contributing to the diminution of the Atrazine-degrading genetic potential in representative soil microsites of earthworm activities. This study illustrates the regulation produced by the earthworms on functional bacterial communities involved in the fate of organic pollutants in soils.     

食肉目哺乳动物的系统发育学研究概述

追溯生物界不同生物类型的起源及进化关系,即重建生物类群的系统发育树是进化生物学领域中一个十分重要的内容。食肉目哺乳动物位于食物链顶端,很多成员不仅在我国野生动物保护工作中占有重要地位,而且还是研究动物适应性进化遗传机制的重要模式生物。因而,食肉目物种作为物种资源中的一个重要类群,其系统发育学一直是国内外研究的热门课题。构建可靠的食肉目分子系统树,无疑将具有重要的进化理论意义和保护生物学价值。鉴于目前食肉目各科间系统发育关系仍然处于“广泛争论”的状态,本文将针对食肉目科水平上的系统发育学研究进展,包括来自于形态学特征、细胞学及分子生物学方面的证据,做简要概述,并提出目前研究中存在的问题。这对今后食肉目系统发育方面的进一步研究工作具有指导意义,并为以该类群作为模式生物开展适应性进化研究奠定基础。     

斜带石斑鱼囊胚期胚胎和尾芽期胚胎差异表达基因的筛选及克隆

以斜带石斑鱼囊胚期胚胎和尾芽期胚胎分别作为检验组和驱动组,构建了石斑鱼囊胚期胚胎和尾芽期胚胎的抑制性差减杂交cDNA文库。以α-tubulin作为检测指标,显示差减效率分别高达28和27。分别取囊胚期胚胎和尾芽期胚胎各192和960个PCR阳性克隆进行斑点杂交,得到15个囊胚期和131个尾芽期的斑点杂交阳性克隆。测序和数据库比对分析表明,囊胚期15个阳性克隆中有11个已知基因的cDNA片段和没有同源性的4个cDNA片段;而在尾芽期的131个阳性克隆中,有123个已知基因的cDNA片段和8个没有同源性的cD-NA片段。用半定量RT-PCR技术分析了部分基因片段在胚胎发育过程中的表达规律和和组织分布情况。这些差异表达片段的呈现为进一步揭示石斑鱼胚胎发育、早期性别决定和性腺分化的分子机制奠定了基础。     

Characterization and physical mapping of ribosomal RNA gene families in Plantago

• Background and Aims The organization of rRNA genes incultivated Plantago ovata Forsk. and several of its wild allieswas analysed to gain insight into the phylogenetic relationshipsof these species in the genus which includes some 200 species. • Methods Specific primers were designed to amplify theinternal transcribed spacer (ITS1 and ITS2) regions from sevenPlantago species and the resulting fragments were cloned andsequenced. Similarly, using specific primers, the 5S rRNA genesfrom these species were amplified and subsequently cloned. Fluorescencein-situ hybridization (FISH) was used for physical mapping of5S and 45S ribosomal RNA genes. • Results The ITS1 region is 19–29 bp longer thanthe ITS2 in different Plantago species. The 5S rRNA gene-repeatingunit varies in length from 289 to 581 bp. Coding regions arehighly conserved across species, but the non-transcribed spacers(NTS) do not match any database sequences. The clone from thecultivated species P. ovata was used for physical mapping ofthese genes by FISH. Four species have one FISH site while threehave two FISH sites. In P. lanceolata and P. rhodosperma, the5S and 45S (18S-5·8S-25S) sites are coupled. • Conclusions Characterization of 5S and 45S rRNA geneshas indicated a possible origin of P. ovata, the only cultivatedspecies of the genus and also the only species with x = 4, froma species belonging to subgenus Psyllium. Based on the studiesreported here, P. ovata is closest to P. arenaria, althoughon the basis of other data the two species have been placedin different subgenera. FISH mapping can be used as an efficienttool to help determine phylogenetic relationships in the genusPlantago and show the interrelationship between P. lanceolataand P. lagopus.     

The ascidian homolog of the vertebrate homeobox gene Rx is essential for ocellus development and function

The tadpole larvae prosencephalon of the ascidian Ciona intestinalis contains a single large ventricle, along the inner walls of which lie two sensory organs: the otolith (a gravity-sensing organ) and the ocellus (a photo-sensing organ composed of a single cup-shaped pigment cell, about 20 photoreceptor cells, and three lens cells). Comparison has been drawn between the morphology and physiology of photoreceptor cells in the ascidian ocellus and the vertebrate eye. The development of vertebrate and invertebrate eyes requires the activity of several conserved genes and it is regulated by precise expression patterns and cell fate decisions common to several species. We have isolated a Ciona homeobox gene (Ci-Rx) that belongs to the paired-like class of homeobox genes. Rx genes have been identified from a variety of organisms and have been demonstrated to have a role in vertebrate eye formation. Ci-Rx is expressed in the anterior neural plate in the middle tailbud stage and subsequently in the larval stage in the sensory vesicle around the ocellus. Loss of Ci-Rx function leads to an ocellus-less phenotype that shows a loss of photosensitive swimming behavior, suggesting the important role played by Ci-Rx in basal chordate photoreceptor cell differentiation and ocellus formation. Furthermore, studies on Ci-Rx regulatory elements electroporated into Ciona embryos using LacZ or GFP as reporter genes indicate the presence of Ci-Rx in pigment cells, photoreceptors, and neurons surrounding the sensory vesicle. In Ci-Rx knocked-down larvae, neither basal swimming activity nor shadow responses develop. Thus, Rx has a role not only in pigment cells and photoreceptor formation but also in the correct development of the neuronal circuit that controls larval photosensitivity and swimming behavior. The results suggest that a Ci-Rx "retinal" territory exists, which consists of pigment cells, photoreceptors, and neurons involved in transducing the photoreceptor signals.     

Identification of Pip4k2β as a mechanical stimulus responsive gene and its expression during musculoskeletal tissue healing

To investigate the mechano-transduction system of cells, we identified genes responsive to a cyclic mechanical stimulus. MC3T3.E1 cells were cultured on a computer-controlled vacuum-pump-operated device designed to provide a cyclic mechanical stimulus. A maximum elongation of 15% of membrane at 10 cycles/min (3 s extension followed by 3 s relax per cycle) was repeated for 48 h. By means of a differential display, the gene expression pattern of cells exposed to the stimulus was compared with that of unexposed cells. As a result, a gene fragment that was exclusively expressed in mechanically stressed cells was identified. By using expressed sequence tag walking together with the oligo-capping method, this gene was identified as phosphatidylinositol 4-phosphate 5-kinase type II β (initially known as Pip5k2β but now reclassified as Pip4k2β). The specific up-regulation of Pip4k2β upon mechanical stimulus was also confirmed by using another apparatus, viz. a computer-controlled linearized-stepping motor system. To examine the involvement of the cyclic mechanical stimulus in the regulation of Pip4k2β expression in musculoskeletal tissue, we created an Achilles tendon transection model in rabbits. The temporal expression of Pip4k2β was assessed by means of a quantitative reverse-transcribed polymerase chain reaction. In the gastrocnemius muscle, expression of Pip4k2β rapidly decreased 1 week after transection but was restored to normal levels at 4 weeks. In the Achilles tendon, however, expression remained decreased until 4 weeks after transection. We suggest that the expression of Pip4k2β can be used as a marker for cells receiving a suitable mechanical stimulus. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.     

Genetic and molecular study of the inability of the yeast Kluyveromyces lactis var. drosophilarum to ferment lactose

The fermentation of lactose (Lac⁺) in the dairy yeast Kluyveromyces lactis var. lactis is controlled by the LAC4 (β-galactosidase) and LAC12 (lactose permease) genes. The complementation analysis of twelve Kl. lactis var. drosophilarum natural homothallic Lac^? strains of different origin was carried out using the genetic heterothallic lines of Kl. lactis var. lactis of the lac4LAC12 and LAC4lac12 genotypes. It was shown that the natural Lac^? strains did not possess the LAC4LAC12 gene cluster. Southern hybridization of chromosomal DNA with LAC4 and LAC12 probes, as well as recombination analysis, showed that Kl. lactis var. drosophilarum yeasts do not have even silent copies of these genes. As distinct from this yeast, natural Lac^? strains of the yeast Kl. marxianus are mutants impaired in the lactose permease gene (lac12 analogue), but possess an active β-galactosidase gene (LAC4 analogue). The origin of the LAC4LAC12 gene cluster of the dairy yeasts Kl. lactis is discussed.