Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping.

Fluorescent hybridization probes were multiplexed for color genotyping of the apolipoprotein E locus using model oligonucleotide targets. Fluorescence resonance energy transfer was observed during adjacent hybridization of 3'-fluorescein-labeled "donor" probes paired with 5'-labeled "acceptor" probes with different emission spectra reporting at codons 112 and 158. The acceptor dyes emitted at either 640 nm (LightCycler Red 640) or 705 nm (LightCycler Red 705) and were monitored with a LightCycler, a thermal cycler with an integrated fluorimeter. The color of the acceptor dye identified each site and the characteristic melting temperatures of the fluorescein-labeled probes identified single base changes within each codon. Color compensation of temperature-dependent spectral overlap was applied to completely separate each channel. Competition between the probes and the complementary strand for the target sequence decreased resonance energy transfer, indicating an advantage of single-stranded target. Hybridization probes of the same length, but different GC content are T(m) shifted by the same amount during A:C mismatch duplex melting. Genotyping was optimal at both sites if melting curve analysis was preceded by a slow (1 degrees C/s) annealing phase. Although each site preferred different concentrations of Mg(2+) and target strand for optimal genotyping, conditions for multiplexing were found. This method, along with an appropriate amplification technique, should allow real-time multiplex genotyping from genomic DNA.     

Mavericks, a novel class of giant transposable elements widespread in eukaryotes and related to DNA viruses

We previously identified a group of atypical mobile elements designated Mavericks from the nematodes Caenorhabditis elegans and C. briggsae and the zebrafish Danio rerio. Here we present the results of comprehensive database searches of the genome sequences available, which reveal that Mavericks are widespread in invertebrates and non-mammalian vertebrates but show a patchy distribution in non-animal species, being present in the fungi Glomus intraradices and Phakopsora pachyrhizi and in several single-celled eukaryotes such as the ciliate Tetrahymena thermophila, the stramenopile Phytophthora infestans and the trichomonad Trichomonas vaginalis, but not detectable in plants. This distribution, together with comparative and phylogenetic analyses of Maverick-encoded proteins, is suggestive of an ancient origin of these elements in eukaryotes followed by lineage-specific losses and/or recurrent episodes of horizontal transmission. In addition, we report that Maverick elements have amplified recently to high copy numbers in T. vaginalis where they now occupy as much as 30% of the genome. Sequence analysis confirms that most Mavericks encode a retroviral-like integrase, but lack other open reading frames typically found in retroelements. Nevertheless, the length and conservation of the target site duplication created upon Maverick insertion (5- or 6-bp) is consistent with a role of the integrase-like protein in the integration of a double-stranded DNA transposition intermediate. Mavericks also display long terminal-inverted repeats but do not contain ORFs similar to proteins encoded by DNA transposons. Instead, Mavericks encode a conserved set of 5 to 9 genes (in addition to the integrase) that are predicted to encode proteins with homology to replication and packaging proteins of some bacteriophages and diverse eukaryotic double-stranded DNA viruses, including a DNA polymerase B homolog and putative capsid proteins. Based on these and other structural similarities, we speculate that Mavericks represent an evolutionary missing link between seemingly disparate invasive DNA elements that include bacteriophages, adenoviruses and eukaryotic linear plasmids.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

The transposable element landscape of the model legume Lotus japonicus

The largest component of plant and animal genomes characterized to date is transposable elements (TEs). The availability of a significant amount of Lotus japonicus genome sequence has permitted for the first time a comprehensive study of the TE landscape in a legume species. Here we report the results of a combined computer-assisted and experimental analysis of the TEs in the 32.4 Mb of finished TAC clones. While computer-assisted analysis facilitated a determination of TE abundance and diversity, the availability of complete TAC sequences permitted identification of full-length TEs, which facilitated the design of tools for genomewide experimental analysis. In addition to containing all TE types found in previously characterized plant genomes, the TE component of L. japonicus contained several surprises. First, it is the second species (after Oryza sativa) found to be rich in Pack-MULEs, with >1000 elements that have captured and amplified gene fragments. In addition, we have identified what appears to be a legume-specific MULE family that was previously identified only in fungal species. Finally, the L. japonicus genome contains many hundreds, perhaps thousands of Sireviruses: Ty1/copia-like elements with an extra ORF. Significantly, several of the L. japonicus Sireviruses have recently amplified and may still be actively transposing.     

Evolution of the Schlafen genes,a gene family associated with embryonic lethality,meiotic drive,immune processes and orthopoxvirus virulence

Genes of the Schlafen family, first discovered in mouse, are expressed in hematopoietic cells and are involved in immune processes. Previous results showed that they are candidate genes for two major phenomena: meiotic drive and embryonic lethality (DDK syndrome). However, these genes remain poorly understood, mostly due to the limitations imposed by their similarity, close location and the potential functional redundancy of the gene family members.     

The limited distribution of Helitrons to vesper bats supports horizontal transfer

Transposable elements (TEs) have the unique ability to move and replicate within the genome and therefore engender dramatic changes to genome architecture. Among different types of TEs, rolling-circle transposons (Helitrons) are well known for their ability to capture and amplify host gene fragments. Bioinformatic analysis revealed that Helitrons constitute ~3% of the Myotis lucifugus, (little brown bat) genome, while no Helitrons were found in any of the other 44+ sequenced mammalian genomes. Recently horizontal transfer has been implicated for some of the M. lucifugus Helitrons, in part explaining this disparate distribution among mammals. The purpose of this work is to determine both the distribution of Helitrons among bats and to estimate the number of independent invasions. We employed a combination of in silico, PCR and hybridization based techniques to identify Helitrons from diverse bat species belonging to ten different families. This work reveals that Helitrons invaded the vesper bat lineage, at least once. Indeed, Helitrons were not identified in the sister taxa 'Miniopterus', which suggests that the amplification of Helibat occurred (30-36 mya) only in the vesper bat lineage. The estimated age of amplification of the Helibats and the rapid radiation of vesper bats are roughly coincidental and suggest that the invasion and amplification of these elements might have influenced their evolutionary trajectory potentially contributing to phenotypic and genotypic diversity.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.