Genetic evidence for a mitochondriate ancestry in the 'amitochondriate' flagellate Trimastix pyriformis

Most modern eukaryotes diverged from a common ancestor that contained the alpha-proteobacterial endosymbiont that gave rise to mitochondria. The 'amitochondriate' anaerobic protist parasites that have been studied to date, such as Giardia and Trichomonas harbor mitochondrion-related organelles, such as mitosomes or hydrogenosomes. Yet there is one remaining group of mitochondrion-lacking flagellates known as the Preaxostyla that could represent a primitive 'pre-mitochondrial' lineage of eukaryotes. To test this hypothesis, we conducted an expressed sequence tag (EST) survey on the preaxostylid flagellate Trimastix pyriformis, a poorly-studied free-living anaerobe. Among the ESTs we detected 19 proteins that, in other eukaryotes, typically function in mitochondria, hydrogenosomes or mitosomes, 12 of which are found exclusively within these organelles. Interestingly, one of the proteins, aconitase, functions in the tricarboxylic acid cycle typical of aerobic mitochondria, whereas others, such as pyruvate:ferredoxin oxidoreductase and [FeFe] hydrogenase, are characteristic of anaerobic hydrogenosomes. Since Trimastix retains genetic evidence of a mitochondriate ancestry, we can now say definitively that all known living eukaryote lineages descend from a common ancestor that had mitochondria.     

Ultrastructure of Trimastix pyriformis (Klebs) Bernard et al.: similarities of Trimastix species with retortamonad and jakobid flagellates.

Trimastix pyriformis (Klebs 1893) Bernard et al. 1999, is a quadriflagellate, free-living, bacterivorous heterotrophic nanoflagellate from anoxic freshwaters that lacks mitochondria. Monoprotist cultures of this species contained naked trophic cells with anterior flagellar insertion and a conspicuous ventral groove. Bacteria were ingested at the posterior end of the ventral groove, but there was no persistent cytopharyngeal complex. The posterior flagellum resided in this groove, and bore two prominent vanes. A Golgi body (dictyosome) was present adjacent to the flagellar insertion. The kinetid consisted of four basal bodies, four microtubular roots, and associated fibers and bands. Duplicated kinetids, each with four basal bodies and microtubular root templates, appeared at the poles of the open mitotic spindle. Trimastix pyriformis is distinguishable from other Trimastix species on the basis of external morphology, kinetid architecture and the distribution of endomembranes. Trimastix species are most similar to jakobid flagellates, especially Malawimonas jakobiformis, and to species of the retortamonad genus Chilomastix. Retortamonads may have evolved from a Trimastix-like ancestor through loss of "canonical" (easily seen with electron microscopy) endomembrane systems and elaboration of cytoskeletal elements associated with the cytostome/cytopharynx complex.     

Evidence of a Cadmium-Thionein and the Glycine Cleavage System in Oxytricha granulifera

ABSTRACT. This work presents the further purification of a Cd-linking protein in Oxytricha granulifera by reverse-phase chromatography. This protein contains 25% cysteine and no aromatic amino acid. it may be considered as a chelatin with some similarity to metallothioneins. During the purification of another Cd-linking compound, we were able to demonstrate that the H protein precursor of glycine cleavage is present in Oxytricha. This is the first finding of the presence of this system in Protozoa.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

Complete sequence of the mitochondrial genome of Tetrahymena pyriformis and comparison with Paramecium aurelia mitochondrial DNA

We report the complete nucleotide sequence of the Tetrahymena pyriformis mitochondrial genome and a comparison of its gene content and organization with that of Paramecium aurelia mtDNA. T. pyriformis mtDNA is a linear molecule of 47,172 bp (78.7 % A+T) excluding telomeric sequences (identical tandem repeats of 31 bp at each end of the genome). In addition to genes encoding the previously described bipartite small and large subunit rRNAs, the T. pyriformis mitochondrial genome contains 21 protein-coding genes that are clearly homologous to genes of defined function in other mtDNAs, including one (yejR) that specifies a component of a cytochrome c biogenesis pathway. As well, T. pyriformis mtDNA contains 22 open reading frames of unknown function larger than 60 codons, potentially specifying proteins ranging in size from 74 to 1386 amino acid residues. A total of 13 of these open reading frames (ciliate-specific) are found in P. aurelia mtDNA, whereas the remaining nine appear to be unique to T. pyriformis; however, of the latter, five are positionally equivalent and of similar size in the two ciliate mitochondrial genomes, suggesting they may also be homologous, even though this is not evident from sequence comparisons. Only eight tRNA genes encoding seven distinct tRNAs are found in T. pyriformis mtDNA, formally confirming a long-standing proposal that most T. pyriformis mitochondrial tRNAs are nucleus-encoded species imported from the cytosol. Atypical features of mitochondrial gene organization and expression in T. pyriformis mtDNA include split and rearranged large subunit rRNA genes, as well as a split nad1 gene (encoding subunit 1 of NADH dehydrogenase of respiratory complex I) whose two segments are located on and transcribed from opposite strands, as is also the case in P. aurelia. Gene content and arrangement are very similar in T. pyriformis and P. aurelia mtDNAs, the two differing by a limited number of duplication, inversion and rearrangement events. Phylogenetic analyses using concatenated sequences of several mtDNA-encoded proteins provide high bootstrap support for the monophyly of alveolates (ciliates, dinoflagellates and apicomplexans) and slime molds.     

The Properties of an Enzyme System Degrading Endogenous Phospholipids of Tetrahymena pyriformis

SYNOPSIS. Tetrahymena pyriformis contains a potent system of lipolytic enzymes which is activated by disruption of the cells. The extent to which endogenous lipids of the homogenate are degraded depends upon the age of the culture and the strain of T. pyriformis used. In their most active form, the enzymes can hydrolyze nearly 60% of the endogenous phospholipids within one hour at room temperature. The products of this reaction are probably responsible for the frequently reported instability of Tetrahymena enzyme systems in vitro. The use of inhibitors and the careful choice of culture conditions can reduce lipid degradation to a negligible level.     

The Distribution of Tetrahymena pyriformis1

SYNOPSIS. This paper is a brief account of both amicronucleate and sexually active strains of Tetrahymena pyriformis and their distribution with some comments on their possible evolution.     

The Varicella-Zoster Virus ORFS/L (ORF0) Gene Is Required for Efficient Viral Replication and Contains an Element Involved in DNA Cleavage

The genome of varicella-zoster virus (VZV), a human alphaherpesvirus, consists of two unique regions, unique long (U_L) and unique short (U_S), each of which is flanked by inverted repeats. During replication, four isomers of the viral DNA are generated which are distinguished by the relative orientations of U_L and U_S. VZV virions predominantly package two isomeric forms of the genome that have a fixed orientation of U_L. An open reading frame (ORF) of unknown function, ORFS/L, also referred to as ORF0, is located at the extreme terminus of U_L, directly adjacent to the a-like sequences, which are known to be involved in cleavage and packaging of viral DNA. We demonstrate here that the ORFS/L protein localizes to the Golgi network in infected and transfected cells. Furthermore, we were able to demonstrate that deletion of the predicted ORFS/L gene is lethal, while retention of the N-terminal 28 amino acid residues resulted in viable yet replication-impaired virus. The growth defect was only partially attributable to the expression of the ORFS/L product, suggesting that the 5′ region of ORFS/L contains a sequence element crucial for cleavage/packaging of viral DNA. Consequently, mutations introduced into the extreme 5′ terminus of ORFS/L resulted in a defect in DNA cleavage, indicating that the region is indeed involved in the processing of viral DNA. Since the sequence element has no counterpart at the other end of U_L, we concluded that our results can provide an explanation for the almost exclusive orientation of the U_L seen in packaged VZV DNA.Varicella-zoster virus ([VZV] Human Herpesvirus 3), is a highly cell-associated alphaherpesvirus that causes chicken pox (varicella) upon infection of naïve individuals (2). During primary infection, VZV is able to establish latency in cranial nerves, as well as dorsal root and autonomic ganglia, where it remains dormant until a reactivation event occurs (11). Reactivation of VZV occurs primarily in elderly or immunocompromised individuals and results in the development of shingles (herpes zoster), which is often associated with severe pain and postherpetic neuralgia (1).The VZV genome, the smallest among the human herpesviruses, is approximately 125 kbp in size and encodes at least 70 unique open reading frames (ORFs) (1). As has been reported for all alphaherpesviruses, the VZV genome consists of two unique regions, unique long (U_L) and unique short (U_S), each flanked by inverted repeat regions (TR_L, IR_L, TR_S, and IR_S) (9). In contrast to herpes simplex virus type 1 (HSV-1), the prototype alphaherpesvirus, VZV contains only very short repeats (88 bp) on either end of U_L, characteristic of members of the Varicellovirus genus (6). During alphaherpesvirus replication, four isomers of viral DNA are generated which can be distinguished by the orientation of U_L and U_S relative to each other. While all four possible isomers of HSV-1 DNA are packaged in virions as equimolar populations, virions produced by VZV and other varicelloviruses, such as equine herpesvirus type 1 (EHV-1), contain predominantly only two of the four possible isomeric forms of the genome (6, 10, 12, 15, 23). It was shown by Southern blot analysis of VZV virion DNA that inversion of the U_L region is rare and occurs in only approximately 5% of cases (6), which also may be attributed to a rare circular configuration of the genome within the virion (14). A previous report on EHV-1 suggested that inversion of the U_L region in infected cells is common but that packaging occurs in a directional manner (23). For both VZV and EHV-1, the reason for the more-or-less exclusive orientation of U_L within the virion still remains unknown.The organization of the VZV genome is similar to that of HSV-1, and over 90% of the VZV ORFs have counterparts in the HSV-1 genome (1, 13). One of the genes with a predicted HSV-1 homologue is ORFS/L, also referred to as ORF0. ORFS/L is predicted to encode a tail-anchored 157-amino-acid (aa) residue type 2 transmembrane protein and was discovered by Kemble and coworkers (13). The gene is located at the very beginning of U_L, directly adjacent to the a-like sequences that contain PacI and PacII sites crucial for the cleavage and packaging of concatameric VZV DNA (Fig. ​(Fig.1)1) (13, 20). Although no function has yet been attributed to the ORFS/L (ORF0) gene or its product, bioinformatic analysis of the VZV genome indicated that it represents a homologue of HSV-1 UL56 (RefSeq accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_001348","term_id":"9625875","term_text":"NC_001348"}}NC_001348) (7, 8). While UL56 is dispensable for HSV-1 replication in vitro, it plays an important role in pathogenicity in vivo (3, 21). Little is known about the molecular mechanism of UL56 function in the case of HSV-1, but UL56 orthologues are specified by most members of the Alphaherpesvirinae subfamily (26). It was shown that the HSV-2 UL56 product localizes to the Golgi network and interacts with KIF1A, a kinesin motor protein, suggesting a role in vesicular trafficking (16, 17).Open in a separate windowFIG. 1.Overview of the VZV ORFS/L genomic region and the mutants generated. (A) Schematic representation of the VZV genome with a focus on the terminal region containing ORFS/L. Scale bars provide an accurate measure of the genome and the expanded region. (B) Overview of the mutants generated with mutations in the ORFS/L region. A cross indicates the deletion of the corresponding region. Black arrows indicate the loci of stop codon or HA tag insertion.A previous study of Kemble and coworkers also addressed the localization of the ORFS/L protein using a rabbit polyclonal antibody. It was reported that the ORFS/L product was found exclusively in the cytoplasm, which is contradictory to the findings for the HSV-2 orthologue and also to the localization of the ORFS/L protein based on in silico predictions from the primary sequence (13). ORFS/L of the P-Oka strain was recently shown to be unglycosylated but present in the virion (18). Furthermore, ORFS/L expression was detected in skin lesions of individuals, as well as neurons of dorsal root ganglia, during virus reactivation (13). In addition, the deletion of aa 29 to aa 157 of ORFS/L was shown to have an effect on viral replication in vitro and in vivo in the SCID-hu mouse model with thymus-liver implants. In this study, a virus-encoded luciferase reporter system was used to evaluate the growth properties of several bacterial artificial chromosome (BAC)-derived VZV mutants (28). However, it has remained unknown whether the observed growth defect is dependent on ORFS/L gene function or is due to the deletion of another critical sequence element.In this study, we sought to perform a systematic analysis of ORFS/L sequences. We were able to demonstrate that the ORFS/L protein localizes to the Golgi network in infected and transfected cells, providing further evidence for its predicted structure as a tail-anchored type 2 transmembrane protein and lending further support to the notion that it is the orthologue of HSV UL56. In addition, we showed that the ORFS/L gene product is important for efficient VZV replication in vitro. However, we also identified a 5′ region of the predicted ORFS/L that is essential for replication and plays a role in cleavage of viral DNA, as previously suggested by Davison and colleagues (6, 7). Since this essential region is not present at the opposite end of U_L, it could provide an explanation for the almost exclusive packaging in VZV virions of two viral DNA isomers with an invariable U_L orientation.     

The Amount of Metabolizable Glycine

Two roosters of Single Comb White Leghorn breed were fed on a formula feed containing 2-¹⁴C glycine for 16~17 days. The animals were killed and the specific activities of glycine in the tissue proteins and the purine ring of the excreted uric acid were measured. The amount of synthesized glycine in the rooster was calculated by the dilution method based on the specific activities of glycine in the liver protein and the absorbed one. The rooster absorbed about 1 g of glycine and synthesized about 10 g of glycine per day. Quantitative aspect of glycine metabolism in the rooster was discussed.     

The macronuclear polyubiquitin gene of the ciliate Tetrahymena pyriformis.

The presence of ubiquitin in ciliates was first demonstrated in Tetrahymena pyriformis. One clone--pTU2--presents two incomplete open reading frames and the putative polyubiquitin genes have been shown to be highly similar to those of other organisms. To further analyze the organization of this multigene family, several fragments of macronuclear DNA were cloned. We report here the isolation and characterization of one genomic clone (pTU20) that encodes a polyubiquitin gene (TU20) with five tandem repeats and presenting only one extra triplet CAA (Gln) upstream from the TGA. The promoter region of TU20 also presents a consensus heat shock element. The specific detection of RNA species with a synthetic oligonucleotide probe reveals that it corresponds to the 1.8 kb mRNA species whose expression is increased by temperature stress.     

IcmQ in the Type 4b Secretion System Contains an NAD+ Binding Domain

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