Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

A method for the isolation of genomic fragments of RNA virus based on cDNA representational difference analysis (cDNA RDA) was developed. cDNA RDA has been applied for the subtraction of poly(A)(+) RNAs but not for poly(A)(−) RNAs, such as RNA virus genomes, owing to the vast quantity of ribosomal RNAs. We constructed primers for inefficient reverse transcription of ribosomal sequences based on the distribution analysis of hexanucleotide patterns in ribosomal RNA. The analysis revealed that distributions of hexanucleotide patterns in ribosomal RNA and virus genome were different. We constructed 96 hexanucleotides (non-ribosomal hexanucleotides) and used them as mixed primers for reverse transcription of cDNA RDA. A synchronous analysis of hexanucleotide patterns in known viral sequences showed that all the known genomic-size viral sequences include non-ribosomal hexanucleotides. In a model experiment, when non-ribosomal hexanucleotides were used as primers, in vitro transcribed plasmid RNA was efficiently reverse transcribed when compared with ribosomal RNA of rat cells. Using non-ribosomal primers, the cDNA fragments of severe acute respiratory syndrome coronavirus and bovine parainfluenza virus 3 were efficiently amplified by subtracting the cDNA amplicons derived from uninfected cells from those that were derived from virus-infected cells. The results suggest that cDNA RDA with non-ribosomal primers can be used for species-independent detection of viruses, including new viruses

The control of expression of storage protein genes in pisum sativum L

Pea cotyledon and leaf genomic DNA were found to be methylated in a series of defined methylation states. ͫ CG and ͫ C-X-G methylations were detected and the latter was more prevalent in leaf DNA. Pea rDNA was also found to be highly methylated but was relatively undermethylated in the developing cotyledon. The significance of the relative hypo-' methylation of cotyledon genomic DNA (and rDNA) is discussed with respect to the endoreduplication phase of seed development. Two post-expression demethylation events associated with the legumin gene family were detected using a cDNA probe. The methylation of specific CCGG sequences in and around two legumin genes was also investigated. The extent of the methylation of the genes was found to increase in a 5' to 3' direction and one gene was found to have an unmethylated site about 500bp upstream from the transcription start site. Minor changes in the extent of methylation of two sites in the protein coding regions of the two genes were detected and these are thought to represent 'fine-tuning' of gene expression, rather than major gene switching events. One or two post-expression demethylation events associated with the vicilin gene family, were detected using cDNA probes. In addition, there was evidence that some cytosines associated with the vicilin genes became hypermethylated during cotyledon development. A normal pattern of 50,000-M vicilin gene demethylation and hypermethylation was detected in the cotyledon DNA of a mutant pea line, which produces reduced levels of 50,000-M vicilin polypeptide and message.Analysis of the sequence data of two legumin genes indicated that in general the CG dinucleotide was suppressed although one exon was found to have a cluster of CG dinucleotides and an increased usage of the CG-containing arginine codons. The mutability of 5-methylcytosine is discussed in relation to possible legumin protein coding requirements

Developmental cycle, transcriptome and metabolic features of the chlamydial symbiont Protochlamydia amoebophila

Ziel dieser Arbeit war ein besseres Verständnis der Biologie und der möglichen Pathogenität der vor etwa 10 Jahren entdeckten und weitverbreiteten Chlamydien-ähnlichen Bakterien. Mittels eines hochempfindlichen PCR-basierten Screenings von Proben aus Patienten mit Lungenentzündung konnte DNA von drei verschiedenen Chlamydiensymbionten nachgewiesen werden. Ob diese Bakterien, die zum ersten Mal in humanen respiratorischen Proben detektiert wurden, tatsächlich krankheitsverursachend sind muss in weiteren Untersuchungen geklärt werden. Diese Arbeit beschreibt des Weiteren den viertägigen Entwicklungszyklus von Protochlamydia amoebophila in zwei unterschiedlichen Acanthamoeba Wirts-Zellen und belegt eine stabile symbiotische Beziehung zwischen Symbiont und Wirt. Eisenmangel führte zu einem abnormen Zellwachstum vergleichbar mit persistenten Formen bei pathogenen Chlamydien. In einer weiteren Studie wurde ein DNA-Mikroarray für die Analyse der Genexpression von P. amoebophila während der Infektion von Amöben entwickelt. Erste Transkriptomanalysen zeigten, dass die Mehrzahl der abgelesenen Gene bei Metabolismus- und Transportvorgängen, bei der Proteinsynthese oder bei der Manipulation des Wirts eine große Rolle spielen. Neben Transkriptomanalysen wurde die metabolische Aktivität von P. amoebophila auch auf Einzelzellebene mittels konfokaler Raman Mikrospektroskopie unter Verwendung einer 13C-markierten Aminosäure untersucht. Mittels dieses neuartigen Verfahrens, das erstmals für die Analyse intrazellulärer Bakterien verwendet wurde, konnte gezeigt werden, dass beide Entwicklungsstadien von P. amoebophila nicht nur intrazellulär Phenylalanin aus dem Cytoplasma der Wirtszelle aufnehmen sondern auch außerhalb der Wirtszelle über einen Zeitraum von drei Wochen, über diesen Zeitraum infektiös bleiben und in der Lage sind außerhalb des Wirtes ihr Membranpotential neu aufzubauen. Diese Befunde widerlegen das Dogma, dass alle Chlamydien außerhalb ihres Wirtes unmittelbar ihre Aktivität verlieren.It was the aim of this thesis to contribute to a better understanding of the biology and the pathogenic potential of Chlamydia-like bacteria, which were first identified in the early 1990s. A PCR-based screening of samples from patients with community-acquired pneumonia resulted in the detection of DNA from three different Chlamydia-like bacteria raising the question whether these Chlamydia-like bacteria are involved in respiratory disease. This thesis describes further the four days lasting developmental cycle of Protochlamydia amoebophila in two different Acanthamoeba hosts and provides evidence for a well-balanced symbiotic interaction between host and symbiont. Iron depletion induced abnormal cell growth most similar to the persistent forms of Chlamydiaceae. A microarray consisting of more than 6,500 probes was developed for gene expression analyses of P. amoebophila during infection of acanthamoebae and initial transcriptome analyses showed that the majority of enzyme transcripts are predicted to be involved in metabolism, protein synthesis, molecule uptake and host-cell manipulation. In addition to the transcriptome analyses, the metabolic activity of P. amoebophila was investigated on a single cell level by stable isotope Raman microspectroscopy using a 13C-labelled amino acid. With this technique, which was used the first time to directly observe metabolic traits of intracellular bacteria, it could be demonstrated that both developmental forms of P. amoebophila not only take up phenylalanine within their amoebal host cells but also consume this amino acid extracellularly for several weeks. This newly discovered feature proves that not all chlamydiae rapidly cease their activity outside their host cells

IDENTIFICATION OF A SET OF FREQUENT DECANUCLEOTIDES IN PLANTS AND IN ANIMALS

We studied the frequency distribution of 1,048,576 oligonucleotides 10 bp long in a sample of 1.961 Mbase of genes from plants, made of 635 sequences extracted from GenBank 71.0, with the aim of detecting transcription control signals. Among all decamers, 3255, or 0.3%, had a frequency 10 times higher than the mean and were subjected to further statistical analysis. For each of the 3255 decamers (parents), we counted the individual frequencies of the 30 decamers (progeny) differing from the parent by one base mutation, and calculated two variance/mean chi-squares for the progeny, with and without the parent decamer. By studying the distribution of the ratio between the two chi-squares we observed that out of 3255 decamers > 10 times frequent than average, 432 had a chi-square ratio > 1.9. In this residual set, which corresponds to < 0.04 per cent of all possible decamers, only 15 known eukaryotic transcription control elements were found; on the other hand, it included 29 decanucleotides that matched with decanucleotides of a set of Drosophila, 24 with a set from mammals, 13 with a set from yeast and four with a set of viruses--all sets identified with the statistical procedures here described. These decanucloetides are highly repetitive and seem to be present throughout all higher organisms, whereas they are uncommon in mammalian viruses