New insights into Plagiogrammaceae (Bacillariophyta) based on multigene phylogenies and morphological characteristics with the description of a new genus and three new species.

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.

New insights into Plagiogrammaceae (Bacillariophyta) based on multigene phylogenies and morphological characteristics with the description of a new genus and three new species.

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.

The small ribosomal subunit from Thermus thermophilus at 4.5 Å resolution: Pattern fittings and the identification of a functional site

The electron density map of the small ribosomal subunit from Thermus thermophilus, constructed at 4.5 Å resolution, shows the recognizable morphology of this particle, as well as structural features that were interpreted as ribosomal RNA and proteins. Unbiased assignments, carried out by quantitative covalent binding of heavy atom compounds at predetermined sites, led to the localization of the surface of the ribosomal protein S13 at a position compatible with previous assignments, whereas the surface of S11 was localized at a distance of about twice its diameter from the site suggested for its center by neutron scattering. Proteins S5 and S7, whose structures have been determined crystallographically, were visually placed in the map with no alterations in their conformations. Regions suitable to host the fold of protein S15 were detected in several positions, all at a significant distance from the location of this protein in the neutron scattering map. Targeting the 16S RNA region, where mRNA docks to allow the formation of the initiation complex by a mercurated mRNA analog, led to the characterization of its vicinity.

UGA suppression by a mutant RNA of the large ribosomal subunit.

A role for rRNA in peptide chain termination was indicated several years ago by isolation of a 168 rRNA (small subunit) mutant of Escherichia coli that suppressed UGA mutations. In this paper, we describe another interesting rRNA mutant, selected as a translational suppressor of the chain-terminating mutant trpA (UGA211) of E. coli. The finding that it suppresses UGA at two positions in trpA and does not suppress the other two termination codons, UAA and UAG, at the same codon positions (or several missense mutations, including UGG, available at one of the two positions) suggests a defect in UGA-specific termination. The suppressor mutation was mapped by plasmid fragment exchanges and in vivo suppression to domain II of the 23S rRNA gene of the rrnB operon. Sequence analysis revealed a single base change of G to A at residue 1093, an almost universally conserved base in a highly conserved region known to have specific interactions with ribosomal proteins, elongation factor G, tRNA in the A-site, and the peptidyltransferase region of 23S rRNA. Several avenues of action of the suppressor mutation are suggested, including altered interactions with release factors, ribosomal protein L11, or 16S rRNA. Regardless of the mechanism, the results indicate that a particular residue in 23S rRNA affects peptide chain termination, specifically in decoding of the UGA termination codon.

Specific PCR based detection of Phytophthora medicaginis using the intergenic spacer region of the ribosomal DNA

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000:1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

THE DOMAINS OF THE CATALYTIC SUBUNIT OF THE EUKARYOTIC RNA DEGRADING EXOSOME, RRP44P, HAVE DISTINCT FUNCTIONS

The exosome is a 3’ to 5’ exoribonuclease complex that consists of ten essential subunits. In the cytoplasm, the exosome degrades mRNA in a general mRNA turnover pathway and in several mRNA surveillance pathways. In the nucleus, the exosome processes RNA precursors to form small, stable, mature RNA species, including rRNA, snRNA, and snoRNA. In addition to processing these RNAs, the nuclear exosome is also involved in degrading aberrantly processed forms of these RNAs, and others, including mRNA. The 3’ to 5’ exoribonuclease activity of the exosome is contributed by the RNB domain of the only catalytically active subunit, Rrp44p, a member of the RNase II family of enzymes. In addition to the RNB domain, Rrp44p consists of three putative RNA binding domains and has an uncharacterized N-terminus, which includes a CR3 region and PIN domain. In an effort to characterize the cellular functions of the domains of Rrp44p, this study identified a second nuclease active site in the PIN domain. Specifically, the PIN domain exhibits endoribonuclease activity in vitro and is essential for exosome function. Further analysis of the nuclease activities of Rrp44p indicate a role for the exoribonuclease activity of Rrp44p in the cytoplasmic and nuclear exosome. This work has also characterized the CR3 region of Rrp44p, a region that has not yet been characterized in any other protein. This region is needed for the majority, if not all, of the cytoplasmic exosome functions as well as for interaction with the exosome. The CR3 region, along with a histidine residue in the N-terminus of Rrp44p, may coordinate a zinc atom. Preliminary evidence supports a role for this coordination in exosome function. Further investigation, however, is needed to determine the molecular dependence of the exosome on the CR3 region of Rrp44p. Despite its initial discovery thirteen years ago, the essential function of Rrp44p, and the exosome, is not yet known. The studies presented here, however, indicate that the essential function of Rrp44p and the exosome is in the nucleus and depends on the nuclease activities of Rrp44p.

The role of specific regions of ribosomal RNAs in translation termination

The ribosome is a molecular machine that produces proteins in a cell. It consists of RNAs (rRNAs) and proteins. The rRNAs have been implicated in various aspects of protein biosynthesis supporting the idea that they function directly in translation. In this study the direct involvement of rRNA in translation termination was hypothesized and both genetic and biochemical strategies were designed to test this hypothesis. As a result, several regions of rRNAs from both ribosomal subunits were implicated in termination. More specifically, the mutation G1093A in an RNA of the large subunit (23S rRNA) and the mutation C1054A in the small subunit RNA (16S rRNA) of the Escherichia coli ribosome, were shown to affect the binding of the proteins that drive termination, RF1 and RF2. These mutations also caused defects in catalysis of peptidyl-tRNA hydrolysis, the last step of termination. Furthermore, the mutations affected the function of RF2 to a greater extent than that of RF1, a striking result considering the similarity of the RFs. The major defect in RF2 function was consistent with in vivo characteristics of the mutants and can be explained by the inability of the mutant rRNA sites to activate the hydrolytic center, that is the catalytic site for peptidyl-tRNA hydrolysis. Consistent with this explanation is the possibility of a direct interaction between the G1093-region (domain II of 23S rRNA) and the hydrolytic center (most likely domains IV–VI of 23S rRNA). To test that interaction hypothesis selections were performed for mutations in domains IV–VI that compensated for the growth defects caused by G1093A. Several compensatory mutations were isolated which not only restored growth in the presence of G1093A but also appeared to compensate for the termination defects caused by the G1093A. Therefore these results provided genetic evidence for an intramolecular interaction that might lead to peptidyl-tRNA hydrolysis. Finally, a new approach to the study of rRNA involvement in termination was designed. By screening a library of rRNA fragments, a fragment of the 23S rRNA (nt 74-136) was identified that caused readthrough of UGA. The antisense RNA fragment produced a similar effect. The data implicated the corresponding segment of intact 23S rRNA in termination. ^

Haplotype distribution of five nuclear genes based on network genealogies and Bayesian inference indicates that Trypanosoma cruzi hybrid strains are polyphyletic

Chagas disease is still a major public health problem in Latin America. Its causative agent, Trypanosoma cruzi, can be typed into three major groups, T. cruzi I, T. cruzi II and hybrids. These groups each have specific genetic characteristics and epidemiological distributions. Several highly virulent strains are found in the hybrid group; their origin is still a matter of debate. The null hypothesis is that the hybrids are of polyphyletic origin, evolving independently from various hybridization events. The alternative hypothesis is that all extant hybrid strains originated from a single hybridization event. We sequenced both alleles of genes encoding EF-1 alpha, actin and SSU rDNA of 26 T. cruzi strains and DHFR-TS and TR of 12 strains. This information was used for network genealogy analysis and Bayesian phylogenies. We found T. cruzi I and T. cruzi II to be monophyletic and that all hybrids had different combinations of T. cruzi I and T. cruzi II haplotypes plus hybrid-specific haplotypes. Bootstrap values (networks) and posterior probabilities (Bayesian phylogenies) of clades supporting the monophyly of hybrids were far below the 95% confidence interval, indicating that the hybrid group is polyphyletic. We hypothesize that T. cruzi I and T. cruzi II are two different species and that the hybrids are extant representatives of independent events of genome hybridization, which sporadically have sufficient fitness to impact on the epidemiology of Chagas disease.

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromosome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively. (C) 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea : Trematoda : Platyhelminthes)

Phylogenies of trematodes based on characters derived from morphology and life cycles have been controversial. Here, we add molecular data to the phylogenetic study of a group of trematodes, members of the superfamily Hemiuroidea Looss, 1899. DNA sequences from the V4 domain of the nuclear small subunit (18S) rRNA gene and a matrix of morphological characters modified from a previous study were used. There was no significant incongruence between the molecular and the morphological data. However, this was probably due largely to the limited resolving power of the morphological data. Analyses support a monophyletic Hemiuroidea containing at least the families Accacoeliidae, Derogenidae, Didymozoidae, Hirudinellidae, Sclerodistomidae, Syncoeliidae, Isoparorchiidae, Lecithasteridae, and Hemiuridae. These families fall into two principal clades. One contains the first six families and the other the Hemiuridae and lecithasterine lecithasterids. The positions of the hysterolecithine lecithasterids and the Isoparorchiidae were poorly resolved. The Ptychogonimidae may be the sister group of the remaining Hemiuroidea, but there was no support from the molecular data for the placement of the Azygiidae within the superfamily. (C) 1998 Academic Press.

Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes

Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14415 bp), S. japonicum (Anhui strain, China; 14085 bp) and S. mekongi (Khong Island, Laos; 14072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 an), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79 for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species. (C) 2001 Elsevier Science B.V. All rights reserved.

Persistent inhibition of FLIP(L) expression by lentiviral small hairpin RNA delivery restores death-receptor-induced apoptosis in neuroblastoma cells.

Neuroblastoma represents the most common and deadly solid tumour of childhood, which disparate biological and clinical behaviour can be explained by differential regulation of apoptosis. To understand mechanisms underlying death resistance in neuroblastoma cells, we developed small hairpin of RNA produced by lentiviral vectors as tools to selectively interfere with FLIP(L), a major negative regulator of death receptor-induced apoptosis. Such tools revealed highly efficient in interfering with FLIP(L) expression and function as they almost completely repressed endogenous and/or exogenously overexpressed FLIP(L) protein and fully reversed FLIP(L)-mediated TRAIL resistance. Moreover, interference with endogenous FLIP(L) and FLIP(S) significantly restored FasL sensitivity in SH-EP neuroblastoma cell line. These results reveal the ability of lentivirus-mediated shRNAs to specifically and persistently interfere with FLIP expression and support involvement of FLIP in the regulation of death receptor-mediated apoptosis in neuroblastoma cells. Combining such tools with other therapeutic modalities may improve treatment of resistant tumours such as neuroblastoma.

Phylogeny of the kinetoplastida: taxonomic problems and insights into the evolution of parasitism

To further investigate phylogeny of kinetoplastid protozoa, the sequences of small subunit (18S) ribosomal RNA of nine bodonid isolates and ten isolates of insect trypanosomatids have been determined. The root of the kinetoplastid tree was attached to the branch of Bodo designis and/or Cruzella marina. The suborder Trypanosomatina appeared as a monophyletic group, while the suborder Bodonina was paraphyletic. Among bodonid lineages, parasitic organisms were intermingled with free-living ones, implying multiple transitions to parasitism and supporting the `vertebrate-first hypothesis'. The tree indicated that the genera Cryptobia and Bodo are artificial taxa. Separation of fish cryptobias and Trypanoplasma borreli as different genera was not supported. In trypanosomatids, the genera Leptomonas and Blastocrithidia were polyphyletic, similar to the genera Herpetomonas and Crithidia and in contrast to the monophyletic genera Trypanosoma and Phytomonas. This analysis has shown that the morphological classification of kinetoplastids does not in general reflect their genetic affinities and needs a revision.

Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification

Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA). Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25), Desmodus rotundus (20), and Nyctinomops laticaudatus (19). Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19), Tadarida brasiliensis (24% = 6/25), and Desmodus rotundus (20% = 4/20). Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%), Artibeus fimbriatus (1/1, 100%), Sturnira lilium (1/3, 33.3%), Myotis levis (2/3, 66.7%)and Diphylla ecaudata (1/2, 50%). PCR products which could indicate the presence of Pneumocystis (21.56%) were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified). This is the first report of detection of Pneumocystis in bats from Brazil.

Influence de l'initiation de la traduction sur le changement programmé du cadre de lecture en -1 responsable de la synthèse des enzymes du virus de l’immunodéficience humaine de type 1

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est responsable du syndrome de l’immunodéficience acquise (SIDA). Il faut identifier de nouvelles cibles pour le développement d’agents anti-VIH-1, car ce virus développe une résistance aux agents présentement utilisés. Notre but est d’approfondir la caractérisation de l’étape du changement de cadre de lecture ribosomique en -1 (déphasage -1) nécessaire à la production du précurseur des enzymes du VIH-1. Ce déphasage est programmé et effectué par une minorité de ribosomes lorsqu’ils traduisent la séquence dite glissante à un endroit spécifique de l’ARN messager (ARNm) pleine-longueur du VIH-1. L’efficacité de déphasage est contrôlée par le signal stimulateur de déphasage (SSF), une tige-boucle irrégulière située en aval de la séquence glissante. La structure du SSF est déroulée lors du passage d’un ribosome, mais elle peut se reformer ensuite. Nous avons montré que des variations de l’initiation de la traduction affectent l’efficacité de déphasage. Nous avons utilisé, dans des cellules Jurkat-T et HEK 293T, un rapporteur bicistronique où les gènes codant pour les luciférases de la Renilla (Rluc) et de la luciole (Fluc) sont séparés par la région de déphasage du VIH-1. La Rluc est produite par tous les ribosomes traduisant l’ARNm rapporteur alors que la Fluc est produite uniquement par les ribosomes effectuant un déphasage. L’initiation de ce rapporteur est coiffe-dépendante, comme pour la majorité des ARNm cellulaires. Nous avons examiné l’effet de trois inhibiteurs de l’initiation et montré que leur présence augmente l’efficacité de déphasage. Nous avons ensuite étudié l’effet de la tige-boucle TAR, qui est présente à l’extrémité 5’ de tous les ARNm du VIH-1. TAR empêche la liaison de la petite sous-unité du ribosome (40S) à l’ARNm et module aussi l’activité de la protéine kinase dépendante de l’ARN double-brin (PKR). L’activation de PKR inhibe l’initiation en phosphorylant le facteur d’initiation eucaryote 2 (eIF2) alors que l’inhibition de PKR a l’effet inverse. Nous avons étudié l’effet de TAR sur la traduction et le déphasage via son effet sur PKR en utilisant TAR en trans ou en cis, mais à une certaine distance de l’extrémité 5’ afin d’éviter l’interférence avec la liaison de la 40S. Nous avons observé qu’une faible concentration de TAR, qui active PKR, augmente l’efficacité de déphasage alors qu’une concentration élevée de TAR, qui inhibe PKR, diminue cette efficacité. Nous avons proposé un modèle où des variations de l’initiation affectent l’efficacité de déphasage en modifiant la distance entre les ribosomes parcourant l’ARNm et, donc, la probabilité qu’ils rencontrent un SSF structuré. Par la suite, nous avons déterminé l’effet de la région 5’ non traduite (UTR) de l’ARNm pleine-longueur du VIH-1 sur l’efficacité de déphasage. Cette 5’UTR contient plusieurs régions structurées, dont TAR à l’extrémité 5’, qui peut interférer avec l’initiation. Cet ARNm a une coiffe permettant une initiation coiffe-dépendante ainsi qu’un site d’entrée interne des ribosomes (IRES), permettant une initiation IRES-dépendante. Nous avons introduit cette 5’UTR, complète ou en partie, comme 5’UTR de notre ARNm rapporteur bicistronique. Nos résultats démontrent que cette 5’UTR complète inhibe l’initiation coiffe dépendante et augmente l’efficacité de déphasage et que ces effets sont dus à la présence de TAR suivie de la tige-boucle Poly(A). Nous avons aussi construit un rapporteur tricistronique où les ribosomes exprimant les luciférases utilisent obligatoirement l’IRES. Nous avons observé que cette initiation par l’IRES est faible et que l’efficacité de déphasage correspondante est également faible. Nous avons formulé une hypothèse pour expliquer cette situation. Nous avons également observé que lorsque les deux modes d’initiation sont disponibles, l’initiation coiffe dépendante est prédominante. Finalement, nous avons étudié l’effet de la protéine virale Tat sur l’initiation de la traduction et sur l’efficacité de déphasage. Nous avons montré qu’elle augmente l’initiation de la traduction et que son effet est plus prononcé lorsque TAR est située à l’extrémité 5’ des ARNm. Nous proposons un modèle expliquant les effets de Tat sur l’initiation de la traduction par l’inhibition de PKR ainsi que par des changements de l’expression de protéines cellulaires déroulant TAR. Ces résultats permettent de mieux comprendre les mécanismes régissant le déphasage du VIH-1, ce qui est essentiel pour le développement d’agents anti-déphasage.