TSSi--an R package for transcription start site identification from 5' mRNA tag data.

UNLABELLED: High-throughput sequencing has become an essential experimental approach for the investigation of transcriptional mechanisms. For some applications like ChIP-seq, several approaches for the prediction of peak locations exist. However, these methods are not designed for the identification of transcription start sites (TSSs) because such datasets contain qualitatively different noise. In this application note, the R package TSSi is presented which provides a heuristic framework for the identification of TSSs based on 5' mRNA tag data. Probabilistic assumptions for the distribution of the data, i.e. for the observed positions of the mapped reads, as well as for systematic errors, i.e. for reads which map closely but not exactly to a real TSS, are made and can be adapted by the user. The framework also comprises a regularization procedure which can be applied as a preprocessing step to decrease the noise and thereby reduce the number of false predictions. AVAILABILITY: The R package TSSi is available from the Bioconductor web site: www.bioconductor.org/packages/release/bioc/html/TSSi.html.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Visualization of a cytoskeleton-like FtsZ network in chloroplasts.

It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton. Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element. Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure. Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner. Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division. As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.

Physcomitrella patens Reute mCherry as a tool for efficient crossing within and between ecotypes.

Physcomitrella patens is a monoecious moss that is predominantly selfing in the wild. Laboratory crossing techniques have been established and crosses between the sequenced Gransden ecotype and the genetically divergent Villersexel ecotype were used for genetic mapping. The recently introduced ecotype Reute has a high fertility rate and is genetically more closely related to the Gransden ecotype than the Villersexel ecotype. Reute sexual reproduction phenology is similar to Gransden, which should allow successful crossing. Using the Reute ecotype and an existing Gransden mutant as a test case, we applied a normalised crossing approach to demonstrate crossing potential between these ecotypes. Also, using a standard transformation approach, we generated Reute fluorescent strains expressing mCherry that allow an easy detection of crossed offspring (sporophyte). We show that Reute can be successfully crossed with a self-infertile DR5:DsRed2 mutant generated in the Gransden background. Using newly established Reute fluorescent strains, we show that they can efficiently fertilise Reute as well as Gransden wild type. The resulting progeny display Mendelian 1:1 segregation of the fluorescent marker(s), demonstrating the suitability of such strains for genetic crossing. Overall our results demonstrate that Reute is highly suitable for genetic crossing. The Reute mCherry strain can be used as a suitable background for offspring selection after crossing.

Genetic evaluation of fertility traits of dairy cattle using a multiple-trait animal model.

A genetic evaluation system was developed for 5 fertility traits of dairy cattle: interval from first to successful insemination and nonreturn rate to 56 d of heifers, and interval from calving to first insemination, nonreturn rate to 56 d, and interval first to successful insemination of cows. Using the 2 interval traits of cows as components, breeding values for days open were derived. A multiple-trait animal model was applied to evaluate these fertility traits. Fertility traits of later lactations of cows were treated as repeated measurements. Genetic parameters were estimated by REML. Mixed model equations of the genetic evaluation model were solved with preconditioned conjugate gradients or the Gauss-Seidel algorithm and iteration on data techniques. Reliabilities of estimated breeding values were approximated with a multi-trait effective daughter contribution method. Daughter yield deviations and associated effective daughter contributions were calculated with a multiple trait approach. The genetic evaluation software was applied to the insemination data of dairy cattle breeds in Germany, Austria, and Luxembourg, and it was validated with various statistical methods. Genetic trends were validated. Small heritability estimates were obtained for all the fertility traits, ranging from 1% for nonreturn rate of heifers to 4% for interval calving to first insemination. Genetic and environmental correlations were low to moderate among the traits. Notably, unfavorable genetic trends were obtained in all the fertility traits. Moderate to high correlations were found between daughter yield-deviations and estimated breeding values (EBV) for Holstein bulls. Because of much lower heritabilities of the fertility traits, the correlations of daughter yield deviations with EBV were significantly lower than those from production traits and lower than the correlations from type traits and longevity. Fertility EBV were correlated unfavorably with EBV of milk production traits but favorably with udder health and longevity. Integrating fertility traits into a total merit selection index can halt or reverse the decline of fertility and improve the longevity of dairy cattle.

Representation and high-quality annotation of the Physcomitrella patens transcriptome demonstrates a high proportion of proteins involved in metabolism in mosses.

To gain insight into the transcriptome of the well-used plant model system Physcomitrella patens, several EST sequencing projects have been undertaken. We have clustered, assembled, and annotated all publicly available EST and CDS sequences in order to represent the transcriptome of this non-seed plant. Here, we present our fully annotated knowledge resource for the Physcomitrella patens transcriptome, integrating annotation from the production process of the clustered sequences and from a high-quality annotation pipeline developed during this study. Each transcript is represented as an entity containing full annotations and GO term associations. The whole production, filtering, clustering, and annotation process is being modelled and results in seven datasets, representing the annotated Physcomitrella transcriptome from different perspectives. We were able to annotate 63.4 % of the 26 123 virtual transcripts. The transcript archetype, as covered by our clustered data, is compared to a compilation based on all available Physcomitrella full length CDS. The distribution of the gene ontology annotations (GOA) for the virtual transcriptome of Physcomitrella patens demonstrates consistency in the ratios of the core molecular functions among the plant GOA. However, the metabolism subcategory is over-represented in bryophytes as compared to seed plants. This observation can be taken as an indicator for the wealth of alternative metabolic pathways in moss in comparison to spermatophytes. All resources presented in this study have been made available to the scientific community through a suite of user-friendly web interfaces via www.cosmoss.org and form the basis for assembly and annotation of the moss genome, which will be sequenced in 2005.

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes.

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.

The cryptomonad histone H4-encoding gene: structure and chromosomal localization.

Cryptomonads are unicellular flagellates whose plastids are surrounded by four membranes. A periplastidal compartment, containing eukaryote-type ribosomes, starch grains and a so-called nucleomorph, is located between the inner and outer membrane pairs. The nucleomorph has been shown to be the vestigial nucleus of a eukaryotic endosymbiont. In order to obtain more information about the chromatin structure of the nucleomorph and the host nuclear chromosomes, we studied the distribution of the histone, H4. H4 was not detectable in the nucleomorph by immunolocalization, thus supporting earlier findings by Gibbs [In: Wiesner et al. (Eds.), Experimental Phycology 1, 1990, pp. 145-157]. Likewise, no H4 DNA was demonstrable in the nucleomorph by Southern hybridization. Sequence analysis, and Southern and Northern blot data of a cryptomonad gene, H4, indicate an intermediate position for these genes between animals and plants.

Low nitric oxide concentrations in exhaled gas and nasal airways of mammals without paranasal sinuses.

To investigate whether relevant levels of nasal nitric oxide (NO) are produced in the absence of paranasal sinuses, we studied 17 healthy baboons, mammals without any paranasal sinuses. The animals were anesthetized with ketamine hydrochloride and breathed spontaneously. While the baboons breathed through a face mask (mouths closed) connected to a respirator, NO concentrations in exhaled gas were sampled from the expiratory limb and analyzed by chemiluminescence. While the animals were breathing ambient air, nasal gas was sampled via a thin plastic tube and analyzed for NO concentrations by chemiluminescence. Mean NO concentration in the exhaled gas was 1.00 +/- 0.59 parts/billion, and NO release was 4.28 +/- 2.72 nl/min. A NO concentration of 4.79 +/- 2.08 parts/billion was found in the nasal gas (NO release: 7.18 +/- 3.13 nl/min). An age-dependent increase in nasal NO levels was not observed. Exhaled and nasal NO concentrations in baboons were markedly lower than in mammals with paranasal sinuses, suggesting that paranasal sinuses might be an anatomic requirement for production of relevant nasal NO levels.

Volumetric MRI measurements of the tree shrew hippocampus.

Protocols suitable for repeated magnetic resonance imaging (MRI) studies of the tree shrew's brain were established. This included the development of (i) a technique for prolonged inhalation anesthesia by endotracheal intubation; (ii) a reproducible fixation of the animal's head in a stereotaxic frame and finally (iii) the set-up of the hardware (rf coil) and software (MRI sequences) of the MRI system. The endotracheal intubation as well as the repeated and prolonged anesthesia showed no complications. The in vivo measurements of the tree shrew's hippocampal formation revealed a high reproducibility. Right and left hippocampal volume was determined as 85.2 mm3 +/- 8% and 87.4 mm3 +/- 10%, respectively. The utility of MRI in delineating alterations in brain anatomy was demonstrated in three animals receiving cortisol via the drinking water (5 mg/animal/day). After a 4-week treatment, in two of the three tree shrews a reduction in hippocampal volume was observed. Thus, the MRI protocols used here allow for repeated and non-invasive measurements of changes in hippocampal anatomy within the same animal and to monitor the temporal dynamics of structural alterations within this brain structure.

The Caenorhabditis elegans GARP complex contains the conserved Vps51 subunit and is required to maintain lysosomal morphology.

In yeast the Golgi-associated retrograde protein (GARP) complex is required for tethering of endosome-derived transport vesicles to the late Golgi. It consists of four subunits--Vps51p, Vps52p, Vps53p, and Vps54p--and shares similarities with other multimeric tethering complexes, such as the conserved oligomeric Golgi (COG) and the exocyst complex. Here we report the functional characterization of the GARP complex in the nematode Caenorhabditis elegans. Furthermore, we identified the C. elegans Vps51 subunit, which is conserved in all eukaryotes. GARP mutants are viable but show lysosomal defects. We show that GARP subunits bind specific sets of Golgi SNAREs within the yeast two-hybrid system. This suggests that the C. elegans GARP complex also facilitates tethering as well as SNARE complex assembly at the Golgi. The GARP and COG tethering complexes may have overlapping functions for retrograde endosome-to-Golgi retrieval, since loss of both complexes leads to a synthetic lethal phenotype.

The SecY protein family: comparative analysis and phylogenetic relationships.

We have cloned and sequenced the plastome encoded secY homologue of the cryptomonad alga Pyrenomonas salina. In this study we have carried out a comparative analysis of all but one fully sequenced proteins of the SecY family. We present an alignment of 16 SecY family proteins, containing family signatures, putative transmembrane domains, consensus sequence, and conservation grade. A phylogenetic tree derived from the conserved blocks of the alignment reveals the relationships among this protein family. The tree shows division into two broad subfamilies, one consisting of prokaryotic and plastidal sequences and the other of eukaryotic as well as archaeal sequences.

Phylogenetic analysis of the stress-70 protein family.

The eukaryotic cyto-/nucleoplasmatic 70-kDa heat-shock protein (HSP70) has homologues in the endoplasmic reticulum as well as in bacteria, mitochondria, and plastids. We selected a representative subset from the large number of sequenced stress-70 family members which covers all known branches of the protein family and calculated and manually improved an alignment. Here we present the consensus sequence of the aligned proteins and putative nuclear localization signals (NLS) in the eukaryotic HSP70 homologues. The phylogenetic relationships of the stress-70 group family members were estimated by use of different computation methods. We present a phylogenetic tree containing all known stress-70 subfamilies and demonstrate the usefulness of stress-70 protein sequences for the estimation of intertaxonic phylogeny.

Alveolar hydatidosis (Echinococcus multilocularis) in a captive rhesus monkey (Macaca mulatta) in Germany.

A naturally occurring E. multilocularis infection in an 11-year-old female rhesus monkey is described. The infection most probably was acquired from foxes of the Göttingen area through contaminated branches or foliage taken from the nearby forest. The most severely affected organs were the liver and the lung, A limited serological survey using the Em2plus ELISA revealed 5 more animals which so far remain clinically healthy as possibly infected.

5HT1A-receptors and behaviour under chronic stress: selective counteraction by testosterone.

Behaviour of chronically stressed male tree shrews is characterized by a reduction in scent marking, self-grooming and overall locomotor activity. It has been proposed that this subordination behaviour is related to the down-regulation of 5HT1A-receptors occurring in distinct brain regions of the animals. The high cortisol concentrations which accompany chronic stress are supposed to induce 5HT1A-receptor down-regulation. Because chronic stress in males also decreases androgen levels we investigated whether behaviour and 5HT1A-receptor expression could be renormalized by testosterone replacement. Male tree shrews were submitted to subordination stress for 28 days, while during the last 18 days, one group was treated with testosterone and one with vehicle. Scent marking, self-grooming, and overall locomotor activity were monitored, and cortisol levels were measured in morning urine during the whole experiment. Brain 5HT1A-receptors were quantified by in vitro receptor autoradiography. Although in subordinate animals cortisol levels remained high during the testosterone treatment, 5HT1A-receptors in the hippocampal formation and the occipital cortex were renormalized to control levels by the androgen, but 5HT1A-receptors in the ventromedial thalamic nucleus did not return to base line levels. Scent marking and self-grooming behaviour were both renormalized by testosterone, but overall locomotor activity did not return to base line levels. These data indicate that a balance between glucocorticoids and androgens is necessary to maintain 'normal' numbers of the monoamine receptors. The fact that both, 5HT1A-receptors and certain behaviours can be renormalized by the sex steroid supports the view that 5HT1A-receptor are involved in the regulation of stress behaviour. However, the fact that overall locomotor activity was not returned to baseline indicates that different types of behaviour are distinctly regulated.

Characterization of a maize ribosomal P2 protein cDNA and phylogenetic analysis of the P1/P2 family of ribosomal proteins.

The nucleotide sequence of a full-length ribosomal P2 protein cDNA from maize was determined and used for a sequence comparison with the P2 and P1 proteins from other organisms. The integration of these data into a phylogenetic tree shows that the P proteins separated into the subspecies P1 and P2 before the eukaryotic kingdoms including plants developed from their ancestor.