Periplast structure of the cryptomonad flagellateHemiselmis brunnescens

Summary The periplast ofHemiselmis brunnescens Butcher is a complex cell covering comprised of the plasma membrane (PM) sandwiched between a surface periplast component (SPC) and an inner periplast component (IPC). The SPC is revealed by deep-etching, and consists of hexagonal plates composed of tripartite subunits that appear to self-assemble into a crystalline layer with a hexagonal symmetry. Small scales (termed fibrillar scales) accumulate on the crystalline plates during cell growth, eventually forming a carpet that itself may appear crystalline when fully formed. Heptagonal rosette scales are occasionally observed on the surface as well. The position of the crystalline plates is precisely mirrored by both the E and P fracture faces of the PM. The plate proper is underlain by membrane with a high concentration of intramembrane particles (IMPs) while the bands of membrane underlying the plate borders lack IMPs. Access of subunits and fibrillar scales to the cell surface following initial plate formation appears to be at the plate boundaries. This study suggests that cryptomonad flagellates may provide model systems for studying the self-assembly of cell surface components, and for relating membrane structure to function, as evidence suggests a major role for the PM in mediating periplast assembly and development.     

Rhodomonas lacustris (Pascher & Ruttner) Javornicky (Cryptomonadida): Ultrastructure of the Vegetative Cell

The ultrastructure of Rhodomonas lacustris was examined using scanning and transmission electron microscopy, and the pigments were scanned in vivo by the opal plate technique. The periplast is composed of hexagonal plates, with trichocysts at the corners. This cryptomonad lacks a gullet but has a deep ventral furrow, in which more than 20 large ejectosomes are located. The chloroplast has mainly three thylakoids per lamella, and it is structurally reminiscent of that of certain haptophycean flagellates. The pigments appear to be as usual within the photosynthetic cryptomonads, with phycoerythrin type PE I (phycocyanin was not detected). Other cellular inclusions (e.g., lysosome-like vesicles and the contractile vacuolar complex) are also discussed.     

The Current Status of the Free-Living Heterotrophic Flagellates

ABSTRACT. The "flagellates" as a taxon has become obsolete and should be dispensed with. It is obsolete because it is not phylo-genetically coherent. Adopting an ultrastructural perspective, it is argued that the flagellates exemplify an extreme case of paraphyly. Meaningful discussion of the evolution or ecology of flagellates can only emerge after we identify the monophyletic constituents of the flagellates. There are over 40 known types of heterotrophic flagellates and these should form the basis of a new understanding of flagellate diversity.     

The Saccharomyces cerevisiae RNase mitochondrial RNA processing is critical for cell cycle progression at the end of mitosis

We have identified a cell cycle delay in Saccharomyces cerevisiae RNase MRP mutants. Mutants delay with large budded cells, dumbbell-shaped nuclei, and extended spindles characteristic of "exit from mitosis" mutants. In accord with this, a RNase MRP mutation can be suppressed by overexpressing the polo-like kinase CDC5 or by deleting the B-type cyclin CLB1, without restoring the MRP-dependent rRNA-processing step. In addition, we identified a series of genetic interactions between RNase MRP mutations and mutations in CDC5, CDC14, CDC15, CLB2, and CLB5. As in most "exit from mitosis" mutants, levels of the Clb2 cyclin were increased. The buildup of Clb2 protein is not the result of a defect in the release of the Cdc14 phosphatase from the nucleolus, but rather the result of an increase in CLB2 mRNA levels. These results indicate a clear role of RNase MRP in cell cycle progression at the end of mitosis. Conservation of this function in humans may explain many of the pleiotropic phenotypes of cartilage hair hypoplasia.     

Phylogenetic relationships of trypanosomatids parasitising true bugs (Insecta: Heteroptera) in sub-Saharan Africa

Three hundred and eighty-six heteropteran specimens belonging to more than 90 species captured in Ghana, Kenya and Ethiopia were examined for the presence of trypanosomatid flagellates. Of those, 100 (26%) specimens were positive for trypanosomatids and the spliced leader RNA gene sequence was obtained from 81 (80%) of the infected bugs. Its sequence-based analysis placed all examined flagellates in 28 typing units. Among 19 newly described typing units, 16 are restricted to sub-Saharan Africa, three belong to previously described species and six to typing units found on other continents. This result was corroborated by the analysis of the ssrRNA gene, sequenced for at least one representative of each major spliced leader RNA-based clade. In all trees obtained, flagellates originating from sub-Saharan Africa were intermingled with those isolated from American, Asian and European hosts, revealing a lack of geographic correlation. They are dispersed throughout most of the known diversity of monoxenous trypanosomatids. However, a complex picture emerged when co-evolution with their heteropteran hosts was taken into account, since some clades are specific for a single host clade, family or even species, whereas other flagellates display a very low host specificity, with a capacity to parasitise heteropteran bugs belonging to different genera/families. The family Reduviidae contains the widest spectrum of trypanosomatids, most likely a consequence of their predatory feeding behaviour, leading to an accumulation of a variety of flagellates from their prey. The plant pathogenic genus Phytomonas is reported here from Africa, to our knowledge for the first time. Finding the same typing units in hosts belonging to different heteropteran families and coming from different continents strongly indicates that the global diversity of the insect trypanosomatids is most likely lower than was predicted on the basis of the "one host-one parasite" paradigm. The analysis presented significantly extends the known diversity of monoxenous insect trypanosomatids and will be instrumental in building a new taxonomy that reflects their true phylogenetic relationships.     

Growth and Reproduction of Leptomonas oncopelti in the Milkweed Bug, Oncopeltus fasciatus

SYNOPSIS. A laboratory colony of Oncopeltus fasciatus was found to be infected with Leptomonas oncopelti. Inasmuch as the parasite is transmitted from parent to offspring an opportunity presented itself to study the biology and transmission of this parasite under controlled laboratory conditions. An apparatus for observing individual bugs was designed and the presence or absence of flagellates in the feces determined. Flagellates were not shed until the bugs became adults after which they appeared in every defecation. Dissection of infected bugs revealed that flagellates were not present in the rectum until adulthood. Further studies indicated that in the midgut of the insect there is a departure from binary fission to budding. The nucleus divides and one of the newly formed nuclei migrates toward a newly formed kinetoplast. Rarely there is still another kinetoplast/nucleus division. In the event the new axoneme grows within the cytoplasmic sheath of the parent flagellum, smaller organisms produced by unequal cytokinesis remain attached. If the axoneme grows free, the smaller daughter organisms become free-swimming. Passage into the rectum of the adult bugs causes a rounding up of all parasites although the leishmaniform organisms continue to divide. It is presumed that infection of clean bugs is accomplished by the ingestion of leishmaniform organisms through a common water source. The reason for the presence of flagellates in the rectum of the adult but not in the nymphal insect and the mechanism responsible for the change from binary to unequal fission are not known.     

Structure and development of the cryptomonad periplast: A review

Summary The structure and development of the complex periplast, or cell covering, of cryptomonads is reviewed. The periplast consists of the plasma membrane (PM) plus an associated surface periplast component (SPC) and cytoplasmic or inner periplast component (IPC). The structure of the SPC and IPC, and their association with the PM, varies considerably between genera. This review, which concentrates on cryptomonads with an IPC of discrete plates, discusses relationships between periplast components and examines the development of this unique cell covering. Formation and growth of inner plates occurs throughout the cell cycle from specialized regions termed anamorphic zones. Crystalline surface plates, which comprise the SPC in many cryptomonad species, appear to form by self-assembly of disorganized subunits. InKomma caudata the subunits are composed of a high molecular weight glycoprotein that is produced within the endomembrane system and deposited onto the cell surface within anamorphic zones. The self-assembly of subunits into highly ordered surface plates appears closely associated with developmental changes in the underlying IPC and PM.     

Coordinate Synthesis but Discrete Localization of Homologous N‐Glycosylated Proteins,CLP and CLB,in Naegleria pringsheimi Flagellates

The synchronous amoebae‐to‐flagellates differentiation of Naegleria pringsheimi has been used as a model system to study the formation of eukaryotic flagella. We cloned two novel genes, Clp, Cl ass I on p lasma membrane and Clb, Cl ass I at b asal bodies, which are transiently expressed during differentiation and characterized their respective protein products. CLP (2,087 amino acids) and CLB (1,952 amino acids) have 82.9% identity in their amino acid sequences and are heavily N‐glycosylated, leading to an ~ 100 × 10³ increase in the relative molecular mass of the native proteins. In spite of these similarities, CLP and CLB were localized to distinct regions: CLP was present on the outer surface of the plasma membrane, whereas CLB was concentrated at a site where the basal bodies are assembled and remained associated with the basal bodies. Oryzalin, a microtubule toxin, inhibited the appearance of CLP on the plasma membrane, but had no effect on the concentration of CLB at its target site. These data suggest that N. pringsheimi uses separate mechanisms to transport CLP and CLB to the plasma membrane and to the site of basal body assembly, respectively.     

Revision of the genus Cryptomonas (Cryptophyceae): a combination of molecular phylogeny and morphology provides insights into a long-hidden dimorphism

Seventy-three strains of cryptophytes assigned to the genera Cryptomonas, Campylomonas or Chilomonas were studied by light microscopy, spectrophotometry and whole-mount electron microscopy. Twelve groups of strains were distinguished by light and whole mount electron microscopy using a combination of characters, mainly cell size, type of periplast and presence/absence and number of pyrenoids. However, characters previously used to distinguish Cryptomonas from Campylomonas (e.g. the type of periplast: polygonal periplast plates vs. a continuous periplast sheet) were found to occur together in dimorphic strains, indicating that periplast types relate to different life-history stages of a single taxon. To evaluate the taxonomic significance of the type of periplast and other characters previously used to distinguish genera and species, representatives of each strain group were subjected to molecular phylogenetic analyses using two nuclear ribosomal DNA regions (ITS2, partial LSU rDNA) and a nucleomorph ribosomal gene (SSU rDNA). The results of the phylogenetic study provide molecular evidence for a life history-dependent dimorphism in the genus Cryptomonas: the genus Campylomonas represents the alternate morph of Cryptomonas. Campylomonas and Chilomonas are reduced to synonyms of Cryptomonas, the genus Cryptomonas is revised and typified, two new species are described and six species are emended.     

The evolutionary transition from subsocial to eusocial behaviour in Dictyoptera: phylogenetic evidence for modification of the "shift-in-dependent-care" hypothesis with a new subsocial cockroach

Cockroaches have always been used to understand the first steps of social evolution in termites because they are close relatives with less complex and integrated social behaviour. Termites are all eusocial and ingroup comparative analysis would be useless to infer the origin of their social behaviour. The cockroach genus Cryptocercus was used as a so-called "prototermite" model because it shows key-attributes similar to the termites (except Termitidae): wood-feeding, intestinal flagellates and subsocial behaviour. In spite of these comparisons between this subsocial cockroach and eusocial termites, the early and remote origin of eusocial behaviour in termites is not well understood yet and the study of other relevant "prototermite" models is however needed. A molecular phylogenetic analysis was carried out to validate a new "prototermite" model, Parasphaeria boleiriana which shows a peculiar combination of these key-attributes. It shows that these attributes of Parasphaeria boleiriana have an independent origin from those of other wood-eating cockroaches and termites. The case of P. boleiriana suggests that a short brood care was selected for with life on an ephemeral wood resource, even with the need for transmission of flagellates. These new phylogenetic insights modify evolutionary hypotheses, contradicting the assumption made with Cryptocercus model that a long brood care is necessary for cooperation between broods in the "shift-in-dependent-care" hypothesis. An ephemeral wood resource is suggested to prompt generation overlap and the evolution of cooperation, even if brood care is shortened.     

B-type cyclins CLB5 and CLB6 control the initiation of recombination and synaptonemal complex formation in yeast meiosis

BACKGROUND: The life cycle of most eukaryotic organisms includes a meiotic phase, in which diploid parental cells produce haploid gametes. During meiosis a single round of DNA replication is followed by two rounds of chromosome segregation. In the first, or reductional, division (meiosis I), which is unique to meiotic cells, homologous chromosomes segregate from one another, whereas in the second, or equational, division (Meiosis II) sister centromeres disjoin. Meiotic DNA replication precedes the initiation of recombination by programmed Spo11-dependent DNA double-strand breaks. Recent reports that meiosis-specific cohesion is established during meiotic S phase and that the length of S phase is modified by recombination factors (Spo11 and Rec8) raise the possibility that replication plays a fundamental role in the recombination process. RESULTS: To address how replication influences the initiation of recombination, we have used mutations in the B-type cyclin genes CLB5 and CLB6, which specifically prevent premeiotic replication in the yeast Saccharomyces cerevisiae. We find that clb5 and clb5 clb6 but not clb6 mutants are defective in DSB induction and prior associated changes in chromatin accessibility, heteroallelic recombination, and SC formation. The severity of these phenotypes in each mutant reflects the extent of replication impairment. CONCLUSIONS: This assemblage of phenotypes reveals roles for CLB5 and CLB6 not only in DNA replication but also in other key events of meiotic prophase. Links between the function of CLB5 and CLB6 in activating meiotic DNA replication and their effects on subsequent events are discussed.     

Observations on Rhinomonas reticulata comb. nov. and R. reticulata var. eleniana var. nov. (Cryptophyceae), with comments on the genera Pyrenomonas and Rhodomonas

Seven marine red strains of Cryptophyceae were examined using scanning and transmission electron microscopy. Six of these, possessing a nucleomorph housed within the pyrenoid and a periplast with distinctly hexagonal periplast areas, are assigned to Rhinomonas reticulata comb. nov. Rhinomonas reticulata var. eleniana var. nov. is described on the basis of the smaller size of the periplast areas. In a strain of the genus Pyrenomonas , which Rhinomonas otherwise resembles, the periplast areas appear more or less rectangular. Since it is impossible to know with certainty which red marine forms fall within the original concept of the genus Rhodomonas , the name Pyrenomonas should be preferred to a recent emendation of Rhodomonas.     

Periplast development in cryptophyceae I. Changes in periplast arrangement throughout the cell cycle

Summary The cell covering (or periplast) of many cryptomonads consists of discrete plate areas precisely arranged over most of the cell periphery. Developmental changes in periplast arrangement that occur throughout the cell cycle are examined here forKomma caudata andProteomonas sulcata [haplomorph]. In both cryptomonads, pole reversal occurs during cytokinesis, necessitating major realignment of the plate areas. Growth of the periplast occurs by addition of new plate areas to specialized regions (termed anamorphic zones) located around the vestibular margins and along the mid-ventral line of cells. Development of the periplast from these regions enables elongation and lateral expansion of cryptomonads throughout cell growth. Observed differences in cell division and periplast development between these genera are closely associated with variations in the arrangement of anamorphic zones.     

Specialization and targeting of B-type cyclins.

The B-type cyclins of S. cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function. We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus. CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p. Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity. Thus, Clb5p may have targeting domains required for CLB5-specific biological activity.     

The surface periplast component of the protistKomma caudata (Cryptophyceae) self-assembles from a secreted high-molecular-mass polypeptide

Summary The cell covering of the cryptomonadKomma caudata (Geitler) Hill is a trilaminar structure consisting of a surface periplast component (SPC) and an inner periplast component (IPC) that sandwich the plasma membrane. In order to investigate the development of the periplast, we have raised monoclonal antibodies against the cell surface ofK. caudata. Immunoblot analyses using one of these antibodies, K1/D.10, showed that it labeled a high-molecular-mass polypeptide. Immunofluorescence and pre- and post-embedding immunogold labeling studies demonstrated that the antibody recognized sites on the cell surface corresponding to the SPC plates and anotherK. caudata cell surface component, the rosulate scales. Labeling was also detected on surface domains devoid of periplast, namely the vestibular/gullet region of the cell. Post-embedding immunocytochemistry revealed that intracellular sites labeled with K1/D.10 included the Golgi apparatus and its associated vesicles. We propose that the subunits of theK. caudata cell covering are antigenically related molecules and that they self-assemble on the cell surface after secretion via the endomembrane system and deployment at the vestibular/gullet region or, in dividing cells, the cytokinetic furrow.     

Gene expression of serine and cysteine proteinase inhibitors during cereal leaf beetle larvae feeding on wheat: the role of insect-associated microorganisms

Plants and insects have been coexisting for more than 350 million years. During this time, both have evolved many strategies to successfully exploit or respond to reciprocal adaptation and defense reactions. Plants tend to minimize the damage caused by pest feeding, while pests tend to manipulate plant response by suppressing plant defense mechanisms or developing strategies to overcome plant defense systems. Plants recognize insect pests by the wounding that they cause and elicitors present in pest oral secretions (saliva and/or regurgitant). These elicitors or insect-associated microorganisms can modulate plant response to the benefit of their insect hosts. In this article, we have undertaken an analysis of gene expression in serine and cysteine proteinase inhibitors (SerPI and CysPI, respectively) in wheat (Triticum aestivum) plants exposed to cereal leaf beetle (CLB, Oulema melanopus, Coleoptera, Chrysomelidae) larvae feeding, and the impact of microbes associated with CLB on the expression levels of these genes. Using three wheat varieties and antibiotic-treated and untreated CLB larvae, we found that SerPI plays a more important role than CysPIs in plant defense against CLB larvae. Additionally, higher levels of SerPI gene expression were observed in systemic leaves in comparison to the wounded leaves (local response). Each of the tested wheat varieties reacted in a specific way to the particular treatment. Moreover, the presence of microbial components associated with insects influenced plant response to CLB larvae feeding.     

RNase MRP cleaves the CLB2 mRNA to promote cell cycle progression: novel method of mRNA degradation

RNase mitochondrial RNA processing (RNase MRP) mutants have been shown to have an exit-from-mitosis defect that is caused by an increase in CLB2 mRNA levels, leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. Here we describe the molecular defect behind this delay. CLB2 mRNA normally disappears rapidly as cells complete mitosis, but the level remains high in RNase MRP mutants. This is in direct contrast to other exit-from-mitosis mutants and is the result of an increase in CLB2 mRNA stability. We found that highly purified RNase MRP cleaved the 5' untranslated region (UTR) of the CLB2 mRNA in several places in an in vitro assay. In vivo, we identified RNase MRP-dependent cleavage products on the CLB2 mRNA that closely matched in vitro products. Disposal of these products was dependent on the 5'-->3' exoribonuclease Xrn1 and not the exosome. Our results demonstrate that the endoribonuclease RNase MRP specifically cleaves the CLB2 mRNA in its 5'-UTR to allow rapid 5' to 3' degradation by the Xrn1 nuclease. Degradation of the CLB2 mRNA by the RNase MRP endonuclease provides a novel way to regulate the cell cycle that complements the protein degradation machinery. In addition, these results denote a new mechanism of mRNA degradation not seen before in the yeast Saccharomyces cerevisiae.     

Cospeciation of termite gut flagellates and their bacterial endosymbionts: Trichonympha species and 'Candidatus Endomicrobium trichonymphae'

Symbiotic flagellates play a major role in the digestion of lignocellulose in the hindgut of lower termites. Many termite gut flagellates harbour a distinct lineage of bacterial endosymbionts, so-called Endomicrobia, which belong to the candidate phylum Termite Group 1. Using an rRNA-based approach, we investigated the phylogeny of Trichonympha , the predominant flagellates in a wide range of termite species, and of their Endomicrobia symbionts. We found that Trichonympha species constitute three well-supported clusters in the Parabasalia tree. Endomicrobia were detected only in the apical lineage (Cluster I), which comprises flagellates present in the termite families Termopsidae and Rhinotermitidae, but apparently absent in the basal lineages (Clusters II and III) consisting of flagellates from other termite families and from the wood-feeding cockroach, Cryptocercus punctulatus . The endosymbionts of Cluster I form a monophyletic group distinct from many other lineages of Endomicrobia and seem to have cospeciated with their flagellate host. The distribution pattern of the symbiotic pairs among different termite species indicates that cospeciation of flagellates and endosymbionts is not simply the result of a spatial separation of the flagellate lineages in different termite species, but that Endomicrobia are inherited among Trichonympha species by vertical transmission. We suggest extending the previously proposed candidatus name ' Endomicrobium trichonymphae ' to all Endomicrobia symbionts of Trichonympha species, and estimate that the acquisition by an ancestor of Trichonympha Cluster I must have occurred about 40–70 million years ago, long after the flagellates entered the termites.     

Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and its biologically active beta-oxidation product phenylacetic acid mustard (PAM; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromatic alkylators. CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant. The chemical structures indicate that CLB and PAM are mutagenic, teratogenic and carcinogenic, but the mode of action has remained obscure. We have investigated the biological effects of CLB and PAM with DNA repair-deficient Escherichia coli strains. In contrast to MNNG (N-methyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E. coli, but permeabilization of the outer membrane of the cells through use of polymyxin B nonapeptide (PMBN) rendered them susceptible to these compounds. The importance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacking O(6)-methylguanine-DNA methyltransferase I and II (ada ogt). Similarly, the protective role of base excision repair (BER) was substantiated by demonstration of an even more increased susceptibility to CLB and PAM of cells lacking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1). Cells deficient in mismatch repair (mutS) appeared to be slightly more sensitive than normal cells to CLB and PAM, although no such sensitivity to MNNG was observed. This implicates the role of mismatches in CLB- and PAM-related cytotoxicity. It is generally believed that bifunctional alkylating agents, like CLB and PAM, exert their cytotoxic action via DNA cross-linking. Our results with O(6)-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient cells indicate that removal of the adducts prior to the formation of cross-links is an important mechanism maintaining cell viability. We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E. coli cells, whose outer membrane is not naturally permeable to mutagens or other interesting compounds.     

应用过氧化物酶同工酶鉴定山茶属植物杂种F1代的研究

对4个杂交组合的12个杂种F1代植株与亲本进行了形态学比较和过氧化物酶同工酶分析,结果表明以金花茶为父本,云南山茶或云南野山茶为母本杂交产生的11个杂种的幼枝、叶的特征均与母本相似,H-86-1-1的花与母本一致,H-87-2-2的花兼有父母本的特征;H-78-1-1的幼枝、叶的特征与父本相似,其花也兼有父母本的特征。有9株的酶谱为“互补型酶带”,且都出现了杂种带,为真正的杂种;其余2株的酶谱与母本一致,与其形态特征表现出一定的相关性,可能为非真正的杂种。同一个杂交组合产生的杂种,酶谱却有差异,金花茶最为特征的一条带(Rf=0.813)在其所有11个杂种F1代植株中都未表达。