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The COBATEST Network of Community-Based Voluntary Counselling and STI/HIV Testing (CBVCT) services was created to standardise monitoring and evaluation of CBVCT services across Europe. This study aims to assess the quality of data collected in the network from 2015 to 2016. A survey was completed by 34 COBATEST Network members and an evaluation was performed of data quality based on three dimensions: transcription validity, completeness and consistency. The weakest area that we identified was data management processes. Only 8.8% of services had a written procedure to address data quality errors, 29.4% had procedures in place to resolve discrepancies and 35.3% performed quality control. We found that 41.2% of services utilised the COBATEST data, 11.8% made decisions based on the COBATEST data and 61.8% analysed their data in an independent manner for internal purposes. We conclude that while services have reliable data to support planning and management of services, improvements to quality procedures would ensure data are translated into evidence. This evidence would support further expansion of CBVCT services in the EU/EEA, including the integration of CBVCT-generated data into national surveillance systems.

This review comprises both well-known and recently described Phytophthora species and concentrates on Phytophthora–woody plant interactions. First, comprehensive data on infection strategies are presented which were the basis for three models that explain invasion and spread of Phytophthora pathogens in different woody host plants. The first model describes infection of roots, the second concentrates on invasion of the trunk, and the last one summarizes infection and invasion of host plants via leaves. On the basis of morphological, physiological, biochemical and molecular data, scenarios are suggested which explain the sequences of reactions that occur in susceptible and tolerant plants following infections of roots or of stem bark. Particular emphasis is paid to the significance of Phytophthora elicitins for such host–pathogen interactions. The overall goal is to shed light on the sequences of pathogenesis to better understand how Phytophthora pathogens harm their host plants. info:eu-repo/semantics/publishedVersion

Telomerase RNA is a subunit of a stable ribonucleoprotein particle required for telomere replication. We find that, at steady state, 5-10% of the telomerase RNA in Saccharomyces cerevisiae and Kluyveromyces lactis contains a poly(A) tail of about 80 nt. In S. cerevisiae, the poly(A)+ fraction quickly disappeared when a conditional pap1 or rna15 mutant was shifted to the nonpermissive temperature, indicating that polyadenylation is accomplished by the same machinery that polyadenylates mRNAs. Potential cis-acting polyadenylation elements were identified in the telomerase RNA sequence; when they were mutagenized, the polyadenylation pattern shifted, but was not eliminated. The corresponding mutants displayed wild-type growth. By putting the RNA under the control of an inducible promoter, we were able to show that synthesis of the poly(A)+ RNA precedes that of the poly(A)- fraction. This supports, but does not prove, a model in which all telomerase RNA is first polyadenylated and then rapidly processed to give the stable poly(A)-form. Cell cycle arrest experiments showed an increase in the poly(A)+ form between G1 and S phase, consistent with an induction of telomerase RNA transcription at the time of DNA replication.

Tertiary interactions are important in the higher-order folding of catalytic RNAs. Recently, a base triple, joining the two major domains of the catalytic core, was determined in group I introns from the cyanobacterium Anabaena PCC7120 and the eukaryote Tetrahymena thermophila. This base triple involves the fifth base pair of P4 and the fifth base of the single-stranded region J8/7. We made base pair and single-nucleotide substitutions in the fifth base pair of P4, a G-C in the wild-type Anabaena intron, and tested them for self-splicing activity. The results suggest a hydrogen bonding model in which only the C of the base pair interacts directly with the fifth base of J8/7. Comparative sequence analysis was used to determine the different combinations of base triples that occur in approximately 450 natural group I introns identified to date. About 94% of the base triples analyzed are compatible with the proposed hydrogen bonding model. Disrupting this base triple in the Tetrahymena intron resulted in the disappearance of splicing intermediates (intron 3' exon and 5' exon), even though the first step of splicing was not affected. Restoration of the base triple by a compensatory mutation reverted the intermediates to wild-type levels. These results suggest that disruption of the base triple increases the rate of the second step of splicing or of a conformational change preceding the second step. Repositioning of the base triple to form a new set of interactions may be required for the second step of splicing.

The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis. It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria. T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA). Each of these tRNAs functions in cytosolic translation. However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well. Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs. Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented. Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import. Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila.

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.

Dimethyl sulfate modification of RNA in living Tetrahymena thermophila allowed assessment of RNA secondary structure and protein association. The self-splicing rRNA intron had the same methylation pattern in vivo as in vitro, indicating that the structures are equivalent and suggesting that this RNA is not stably associated with protein in the nucleolus. Methylation was consistent with the current secondary structure model. Much of telomerase RNA was protected from methylation in vivo, but the A's and C's in the template region were very reactive. Thus, most telomerase is not base paired to telomeres in vivo. Protein-free telomerase RNA adopts a structure different from that in vivo, especially in the template and pseudoknot regions. The U2 snRNA showed methylation protection at the Sm protein-binding sequence and the mRNA branch site recognition sequence. For both telomerase RNA and U2 snRNA, the in vivo methylation pattern corresponded much better to the structure determined by comparative sequence analysis than did the in vitro methylation pattern. Thus, as expected, comparative analysis gives the structure of the RNA in vivo.

The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.

Telomeres, the protective caps of eukaryotic chromosomes, are maintained by the enzyme telomerase. This telomere-specific reverse transcriptase (RT) uses a small region of its RNA subunit as template to synthesize telomeric DNA, which is generally G/T rich in the strand that contains the 3' end. To further our understanding of why telomeres are usually G/T rich, we screened Saccharomyces cerevisiae telomerase RNA (TLC1) libraries with randomized template sequences for complementation of a tlc1 deletion and decapping of existing telomeres. Surprisingly, the vast majority of the 60 000 different mutant telomerase templates tested showed no activity in vivo. This deficiency was not due to impaired assembly with the catalytic subunit (Est2p) nor could it be alleviated by enforced telomerase recruitment to the telomeres. Rather, the mutant templates reduced the nucleotide addition processivity of telomerase. The functional RNA template sequences recovered in our screens preferentially contained two or more consecutive rC nucleotides, reminiscent of the wild-type template. Thus, in contrast to retroviral RTs that can reverse transcribe any RNA sequence into DNA, the budding yeast telomerase RT is specialized for its C-rich RNA template.

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

The past year has seen significant advances in our understanding of telomerase and other factors involved in chromosome end maintenance. The protein subunit of telomerase that provides the active site for telomeric DNA synthesis was identified in ciliates, yeast and mammals. It is structurally related to reverse transcriptase and thus represents the first member of this protein family with an essential cellular function. Telomere DNA-binding proteins that may mediate the interaction of telomerase with telomeres have been identified and further characterized in diverse eukaryotes. A further elucidation of telomeric DNA structure has influenced our view of how telomeres replicate.

Catalytic protein subunits of telomerase from the ciliate Euplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Telomerase, the enzyme that extends chromosomal DNA ends in most eukaryotes, contains essential RNA and protein subunits. We have been studying telomere replication in hypotrichous ciliates such as Euplotes aediculatus, which have numerous short macronuclear DNA molecules and therefore are highly enriched in telomeres and in telomerase. Cloning and sequencing genes for the RNA subunits from several ciliates revealed that telomerase RNAs with insignificant nucleotide sequence homology nevertheless form a common secondary structure. Affinity chromatography based on the sequence of the RNA subunit was used to purify the Euplotes telomerase as an active ribonucleoprotein enzyme. Two protein subunits, 123 kDa and 43 kDa, were identified. The finding of a yeast homologue to the 123 kDa subunit suggests that telomerase protein components may be much more highly conserved in evolution than the RNA subunits. The purified Euplotes telomerase has no activity with blunt-ended DNA primers, but instead requires a four to six nucleotide single-stranded 3' tail. This result supports a model for telomere replication in which other activities such as helicases or nucleases activate replicated DNA for extension by telomerase, a model that may be applicable to telomere replication in diverse eukaryotes.

Synthesis of telomeric repeats at chromosome ends requires telomerase, a ribonucleoprotein enzyme. The RNA subunit, which contains the template for DNA synthesis, has been identified in many organisms. Recently, the protein subunit that catalyzes telomeric DNA extension has also been identified in Euplotes aediculatus and Saccharomyces cerevisiae. It has sequence and functional characteristics of a reverse transcriptase related to retrotransposon and retroviral reverse transcriptases, so this new family of telomerase subunits has been named TRT (Telomerase Reverse Transcriptase). We find it remarkable that the same type of protein structure required for retroviral replication is now seen to be essential for normal chromosome telomere replication in diverse eukaryotes.

Telomerase is the ribonucleoprotein enzyme responsible for the replication of chromosome ends in most eukaryotes. In the ciliate Euplotes aediculatus, the protein p43 biochemically co-purifies with active telomerase and appears to be stoichiometric with both the RNA and the catalytic protein subunit of this telomerase complex. Here we describe cloning of the gene for p43 and present evidence that it is an authentic component of the telomerase holoenzyme. Comparison of the nucleotide sequence of the cloned gene with peptide sequences of the protein suggests that production of full-length p43 relies on a programmed ribosomal frameshift, an extremely rare translational mechanism. Anti-p43 antibodies immunodeplete telomerase RNA and telomerase activity from E.aediculatus nuclear extracts, indicating that the vast majority of mature telomerase complexes in the cell are associated with p43. The sequence of p43 reveals similarity to the La autoantigen, an RNA-binding protein involved in maturation of RNA polymerase III transcripts, and recombinant p43 binds telomerase RNA in vitro. By analogy to other La proteins, p43 may function in chaperoning the assembly and/or facilitating nuclear retention of telomerase.

Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase. Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations. Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity. When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.

Telomerase is a ribonucleoprotein enzyme that uses its internal RNA moiety as a template for synthesis of telomeric repeats at chromosome ends. Here we report the purification of telomerase from Euplotes aediculatus by affinity chromatography with antisense 2'-O-methyl oligonucleotides, a method that was developed for small nuclear ribonucleoprotein particles (snRNPs). Elution of bound ribonucleoprotein from the antisense oligonucleotide under nondenaturing conditions was achieved by a novel approach, using a displacement oligonucleotide. Polypeptides of 120 kDa and 43 kDa (a doublet) copurify with the active telomerase and appear stoichiometric with telomerase RNA. A simple model for DNA end replication predicts that after semiconservative DNA replication, telomerase will extend the newly synthesized, blunt-ended leading strand. We show that purified Euplotes telomerase has no activity with blunt-ended primers. Instead, efficient extension requires 4 to 6 single-stranded nucleotides at the 3' end. Therefore, this model predicts the existence of other activities such as helicases or nucleases that generate a single-stranded 3' end from a blunt end, thus activating the end for telomerase extension.