Variability of a portion of RNA 3 containing the coat protein gene of Citrus variegation virus (CVV) using single-strand conformation polymorphism (SSCP)

A portion of RNA 3 of Citrus variegation virus (CVV), comprising part of the intergenic region and the coat protein (CP) gene from eight viral isolates, was amplified by RT-PCR and cloned. The clones were compared for intra and inter-isolate variations by single-strand conformation polymorphism analysis. Some of the results were compared with sequence data previously obtained. The test discriminated between clones differing in as little as 3.2% of the nucleotides. Most isolates included several variants, in some cases with a predominant pattern, which, however, could no longer be recognised in new RT-PCR products obtained 13 months later. This procedure can there-fore be used to identify and detect variations between CVV isolates. It is rapid, inexpensive and may reduce the amount of sequencing needed for comparing viral isolates

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis

Citrus variegation virus: Molecular variability of a portion of the RNA 3 containing the coat protein gene and design of primers for RT-PCR detection

The coat protein gene and part of the intergenic region of the RNA 3 of several isolates of Citrus variegation virus (CVV) producing either infectious variegation or crinkly leaf symptoms were amplified by RT-PCR, cloned and sequenced. Some isolates were composed of a mixture of sequence variants. The coat protein gene appeared to be highly conserved (lowest similarity among all CVV sequences 93%), especially at the N-terminal, indicating at the molecular level that both types of symptoms are indeed produced by the same virus species. No relationship could be found with the geographic origin. Sequences obtained from isolates producing infectious variegation clustered in a different branch of a dendrogram than those originating from crinkly leaf symptoms. Both clusters could further be distinguished by two parsimonious sites in the coat protein gene. In the short stretch of the intergenic RNA 3 region analysed, a stable hairpin exists in addition to the previously reported hairpin that constitutes the core promoter for the RNA 4 transcription. This second hairpin could also be recognised in the other subgroup 2 Ilarviruses. Surprisingly, at the nucleotide and amino acid levels and in the secondary features of the intergenic region, CVV appeared closer to the other subgroup 2 Ilarviruses than to Citrus leaf rugose virus to which it is serologically related and has been considered to be evolutionary related. Using primers designed for the conserved regions, the virus was detected with a prevalence of 25% and 13% in Portuguese and Moroccan citrus collections. A group of RT-PCR positives was further confirmed by ELISA and biological indexing

Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism