Molecular analysis of the genomic organization of the Hymenoptera Diadromus pulchellus and Eupelmus vuilleti

The genomic organization of two parasitic wasps was analyzed by DNA reassociation. Cot curves revealed a pattern with three types of components. A highly repetitive DNA, accounting for 15 to 25% of the genome, was identified as satellite DNA. The moderately repetitive DNA corresponds to 26 to 42% of the genome in both species, and shows large variations in complexity, repetitive frequency and a number of sub-components between males and females. These variations are seen as resulting from DNA amplification during somatic and sexual differentiation. Dot blot analyses show that such DNA amplifications concern several types of structural and regulatory genes. The presence of repeated mobile elements was studied by the Roninson method to compare the repeated sequence patterns of Diadromus pulchellus and Eupelmus vuilleti with those of Drosophila melanogaster. The occurrence and organization of mobile elements in these Hymenoptera differ from those of the neighboring order of Diptera. The repetitive and unique components define very large genomes (1 to 3 × 10⁹ base pairs). The genomic organization in Parasitica appears to be an extreme drosophilan type. We propose that the germinal genome of these parasitic wasps is primarily composed of satellite DNA blocks and very long stretches of unique sequences, separated by a few repeated and/or variously deleted, interspersed elements of each mobile element family.     

Mitochondrial rps14 is a transcribed and edited pseudogene in Arabidopsis thaliana

We have isolated and analysed a 2 kb region of the mitochondrial genome of Arabidopsis thaliana (Columbia) showing a high level of nucleotide identity with the mitochondrial (mt) rps14 small-subunit ribosomal protein gene from Oenothera berteriana and Vicia faba, as well as with an open reading frame (ORF) located upstream of the nad3 locus in O. berteriana. The rps14 locus is present as a single copy in the A. thaliana mt genome and has a translational stop codon located near the initiation codon, as well as a deletion of one nucleotide that disturbs the coding sequence. The cloning and sequencing of nine amplified mt rps14 cDNAs clearly demonstrated that this gene is transcribed and that the mRNA precursors are edited at three positions, all involving C-to-U conversions. No editing events changing the stop codon and restoring the correct coding sequence were witnessed within the 9 individual cDNA clones. Therefore, we conclude that the single rps14 sequence of the mitochondrial genome from A. thealiana is in fact a pseudogene that is transcribed and edited but not translated.     

A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue

We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue. We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 10⁶ clones from as little as 1 μg of total RNA.     

The possible role of soluble salts in chemical evolution

Summary A model is proposed for a prebiotic environment in which concentration, condensation, and chemical evolution of biomolecules could have taken place. The main reactions expected of proteins, nucleic acids, lipids, and some of their precursors in this environment are examined.The model is based on our previously developed concept of a fluctuating system in which hydration and dehydration processes take place in a cyclic manner. In the present model, however, high concentrations of soluble salts, such as chlorides and sulfates, are taken into account, whereas previously a more or less salt-free system had been assumed. Thus the preponderance of surfaces of soluble salts is implied, even though sparingly soluble minerals, such as clay minerals or quartz, are also present.During the dehydration stage biomolecules tend to leave the solution and concentrate at certain microenvironments, such as in micelles and aggregates, at the liquid-gas surface and, possibly, at the emerging solid surfaces. Moreover, in these brines, and especially during the last stages of dehydration, high temperatures are attainable, which may enhance certain reactions between the organic molecules, and result in a net increase of condensation over degradation.In the dehydrated state, solid-state condensation and synthesis reactions are possible in which the surface of soluble salts may serve as a catalyst. Several reports in the literature support this hypothesis. Hydration brings about dissolution of the minerals and redistribution of the biomolecules. In such a system, evolutionary processes like those postulated by White (1980) and by Lahav and White (1980) are possible. Moreover, since several soluble salts of known geological occurrence are optically active in their crystalline state, the involvement of the model system in the selection and evolution of chiral organic compounds should also be considered. In addition, organic molecules in the above microenvironments are also expected to undergo selective interactions based on factors such as molecular pattern and chiral recognition and hydrophobicity. The proposed system emphasizes the need to develop the theoretical background and experimental methods for the study of interactions among biomolecules in the presence of high salt concentrations and solid surfaces of soluble salts, as well as interactions between the biomolecules and these surfaces.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.     

柯萨奇病毒B组3型基因组剪切片段的序列分析

柯萨奇病毒B组(Coxsackievirus B,CVB)感染细胞时其基因组RNA存在不稳定现象,但产生机制尚不清楚。本研究将柯萨奇病毒B组3型(CVB3)感染细胞后,利用5′ cDNA末端快速扩增技术(5′ rapid amplification of cDNA ends,5′ RACE)扩增并克隆细胞内CVB3基因组片段,并对每条序列及其5′端的二级结构进行分析。结果获得的20条CVB3基因组片段,长度为 2 067～5 547 bp,片段断端主要分布于2Apro和2C编码区。RNAfold分析显示,这些片段多数在5′断点端形成二级茎-环结构。本研究显示,CVB在宿主细胞感染时可形成大量不完整基因组RNA片段,这些片段可在5′断点端形成局部双链结构,提示片段不是随机产生,可能是RNA酶剪切产物。此发现有助于理解CVB基因组不稳定的机制。     

Contrasting consequences of climate change for migratory geese: Predation,density dependence and carryover effects offset benefits of high‐arctic warming

Climate change is most rapid in the Arctic, posing both benefits and challenges for migratory herbivores. However, population‐dynamic responses to climate change are generally difficult to predict, due to concurrent changes in other trophic levels. Migratory species are also exposed to contrasting climate trends and density regimes over the annual cycle. Thus, determining how climate change impacts their population dynamics requires an understanding of how weather directly or indirectly (through trophic interactions and carryover effects) affects reproduction and survival across migratory stages, while accounting for density dependence. Here, we analyse the overall implications of climate change for a local non‐hunted population of high‐arctic Svalbard barnacle geese, Branta leucopsis, using 28 years of individual‐based data. By identifying the main drivers of reproductive stages (egg production, hatching and fledging) and age‐specific survival rates, we quantify their impact on population growth. Recent climate change in Svalbard enhanced egg production and hatching success through positive effects of advanced spring onset (snow melt) and warmer summers (i.e. earlier vegetation green‐up) respectively. Contrastingly, there was a strong temporal decline in fledging probability due to increased local abundance of the Arctic fox, the main predator. While weather during the non‐breeding season influenced geese through a positive effect of temperature (UK wintering grounds) on adult survival and a positive carryover effect of rainfall (spring stopover site in Norway) on egg production, these covariates showed no temporal trends. However, density‐dependent effects occurred throughout the annual cycle, and the steadily increasing total flyway population size caused negative trends in overwinter survival and carryover effects on egg production. The combination of density‐dependent processes and direct and indirect climate change effects across life history stages appeared to stabilize local population size. Our study emphasizes the need for holistic approaches when studying population‐dynamic responses to global change in migratory species.     

Loop-mediated isothermal amplification assay for the detection of Plasmopara viticola infecting grapes

Grapes downy mildew caused by obligate oomycete plant pathogen Plasmopara viticola is a devastating disease worldwide, resulting in significant yield and quality losses. A field survey was conducted in two major grapes cultivated areas of Tamil Nadu for the incidence of grapevine downy mildew. The disease incidence was 43.42%–76.69%, and the highest disease incidence of 76.69% was observed in the Theni district. Totally eight P. viticola isolates were collected from different places in Coimbatore and Theni districts. These isolates were confirmed through microscopic observation and sequencing of COX 2 gene, and the phylogenetic tree was developed to study their phylogenetic relationship among the isolates which shows 97–100% sequence similarity with other P. viticola isolates and less sequence similarity with Plasmopara species. The loop-mediated isothermal amplification (LAMP) assay was developed based on the CesA4 gene sequence of P. viticola. The assay developed was more sensitive as it detected P. viticola genomic DNA up to 20 fmg. LAMP assay specificity was proved by carrying out the assay with genomic DNA extracted from other Oomycetes and fungal plant pathogens. Finally, LAMP assay was validated by testing seventy-eight grapevine leaf samples collected from seven different locations. LAMP assay showed a positive reaction in sixty-two samples tested out of seventy-eight samples tested. Therefore, the LAMP assay described should helpful for early and specific detection of downy mildew pathogen and help in mitigating disease incidence.