Secondary structure of vertebrate telomerase RNA

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA.     

The snoRNA domain of vertebrate telomerase RNA functions to localize the RNA within the nucleus.

Telomerase RNA is an essential component of the ribonucleoprotein enzyme involved in telomere length maintenance, a process implicated in cellular senescence and cancer. Vertebrate telomerase RNAs contain a box H/ACA snoRNA motif that is not required for telomerase activity in vitro but is essential in vivo. Using the Xenopus oocyte system, we have found that the box H/ACA motif functions in the subcellular localization of telomerase RNA. We have characterized the transport and biogenesis of telomerase RNA by injecting labeled wild-type and variant RNAs into Xenopus oocytes and assaying nucleocytoplasmic distribution, intranuclear localization, modification, and protein binding. Although yeast telomerase RNA shares characteristics of spliceosomal snRNAs, we show that human telomerase RNA is not associated with Sm proteins or efficiently imported into the nucleus. In contrast, the transport properties of vertebrate telomerase RNA resemble those of snoRNAs; telomerase RNA is retained in the nucleus and targeted to nucleoli. Furthermore, both nuclear retention and nucleolar localization depend on the box H/ACA motif. Our findings suggest that the H/ACA motif confers functional localization of vertebrate telomerase RNAs to the nucleus, the compartment where telomeres are synthesized. We have also found that telomerase RNA localizes to Cajal bodies, intranuclear structures where it is thought that assembly of various cellular RNPs takes place. Our results identify the Cajal body as a potential site of telomerase RNP biogenesis.     

Structure and function of vertebrate CMP-sialic acid synthetases

Activation of sugars into nucleotide sugars is critical for their entry into biosynthetic pathways. In eukaryotic cells, the activation of the acidic nine-carbon sugar sialic acid to CMP-sialic acid takes place in the cell nucleus, whereas all other nucleotide sugars are made in the cytoplasm. Molecular cloning of vertebrate CMP-sialic acid synthetases confirmed the nuclear localization and introduced new molecular tools for directly exploring the functional mechanisms of the enzymes, as well as the physiological relevance of their nuclear transport. Although major advances have been made in understanding structure-function relationships and defining elements involved in the nuclear transport, the riddle surrounding the physiological relevance of nuclear localization awaits resolution.     

Ecological guild evolution and the discovery of the world's smallest vertebrate

Living vertebrates vary drastically in body size, yet few taxa reach the extremely minute size of some frogs and teleost fish. Here we describe two new species of diminutive terrestrial frogs from the megadiverse hotspot island of New Guinea, one of which represents the smallest known vertebrate species, attaining an average body size of only 7.7 mm. Both new species are members of the recently described genus Paedophryne, the four species of which are all among the ten smallest known frog species, making Paedophryne the most diminutive genus of anurans. This discovery highlights intriguing ecological similarities among the numerous independent origins of diminutive anurans, suggesting that minute frogs are not mere oddities, but represent a previously unrecognized ecological guild.     

Homologs of CD83 from elasmobranch and teleost fish

Dendritic cells are one of the most important cell types connecting innate and adaptive immunity, but very little is known about their evolutionary origins. To begin to study dendritic cells from lower vertebrates, we isolated and characterized CD83 from the nurse shark (Ginglymostoma cirratum (Gici)) and rainbow trout (Oncorhynchus mykiss (Onmy)). The open reading frames for Gici-CD83 (194 aa) and Onmy-CD83 (218 aa) display approximately 28-32% identity to mammalian CD83 with the presence of two conserved N-linked glycosylation sites. Identical with mammalian CD83 genes, Gici-CD83 is composed of five exons including conservation of phase for the splice sites. Mammalian CD83 genes contain a split Ig superfamily V domain that represents a unique sequence feature for CD83 genes, a feature conserved in both Gici- and Onmy-CD83. Gici-CD83 and Onmy-CD83 are not linked to the MHC, an attribute shared with mouse but not human CD83. Gici-CD83 is expressed rather ubiquitously with highest levels in the epigonal tissue, a primary site for lymphopoiesis in the nurse shark, whereas Onmy-CD83 mRNA expression largely paralleled that of MHC class II but at lower levels. Finally, Onmy-CD83 gene expression is up-regulated in virus-infected trout, and the promoter is responsive to trout IFN regulatory factor-1. These results suggest that the role of CD83, an adhesion molecule for cell-mediated immunity, has been conserved over 450 million years of vertebrate evolution.     

The distribution of capillaries in the somatic musculature of two vertebrate types with particular reference to teleost fish

The distribution of capillaries in teleost and rat striated muscles was investigated using a number of different methods. A new method for directly viewing capillaries was developed. Teleost white muscle has a capillary: fibre (C:F) ratio of between 0.2 and 0.3; and 0.6 to 1.0 peripheral capillaries per muscle fibre. 26-49% of fibres had no peripheral capillaries. Values for the rat gastrocnemius were 1.2, 2.6 and 4.8% respectively which compares well with literature values. Flathead red muscle had a C:F ratio of between 1.9 and 2.5; and between 5.3 and 6.6 peripheral capillaries per muscle fibre depending on the method used. Values for rat soleus were 1.8 and 4.1 respectively. Teleost pink fibres had an intermediate number of capillaries. Rat striated muscle, particularly the gastrocnemius, was found to be heterogeneous with respect to the distribution of capillaries. Flathead red muscle was homogeneous whilst teleost white muscle was only slightly variable. Flathead red muscle fibres are well suppled with subsarcolemmal mitochondria. These show a clumped distribution corresponding to the position of capillaries. In contrast teleost white fibres are almost totally devoid of these and all other mitochondria. No differences were observed in the vascularisation of either muscle type along the length of the fish. The results are discussed in relation to the division of labour between fibre types during swimming.     

Towards brain repair: Insights from teleost fish

In the adult mammalian brain, the ability to minimize secondary cell death after injury, and to repair nervous tissue through generation of new neurons, is severely compromised. By contrast, certain taxa of non-mammalian vertebrates possess an enormous potential for regeneration. Examination of one of these taxa, teleost fish, has revealed a close link between this phenomenon and constitutive adult neurogenesis. Key factors mediating successful regeneration appear to be: elimination of damaged cells by apoptosis, instead of necrosis; activation of mechanisms that prevent the occurrence of secondary cell death; increased production of new neurons that replace neurons lost to injury; and activation of developmental mechanisms that mediate directed migration of the new cells to the site of injury, the differentiation of the young cells, and their integration into the existing neural network. Comparative analysis has suggested that constitutive adult neurogenesis is a primitive vertebrate trait, the main function of which has been to ensure a numerical matching between muscle fibers/sensory receptor cells and central elements involved in motor control/processing of sensory information associated with these peripheral elements. It is hypothesized that, when in the course of the evolution of mammals a major shift in the growth pattern from hyperplasia to hypertrophy took place, the number of neurogenic brain regions and new neurons markedly decreased. As a consequence, the potential for neuronal regeneration was greatly reduced, but remnants of neurogenic areas have persisted in the adult mammalian brain in form of quiescent stem cells. It is likely that the study of regeneration-competent taxa will provide important information on how to activate intrinsic mechanisms for successful brain regeneration in humans.     

Cardiac tissue function of the teleost fish Oreochromis niloticus under different thermal conditions

The cardiac responses of Oreochromis niloticus acclimated to 25 degrees C were assessed using ventricle strips mounted for isometric force recording (Fc) and in vivo heart rate (f(H)). f(H) increased progressively from 25 to 40 degrees C. At extracellular Ca(2+) concentrations of 1.25 and 9.25 mM, a transition from 25 to 40 degrees C resulted in a decreased Fc. At both 25 and 40 degrees C, Fc rose when [Ca(2+)] was increased from 1.25 to 9.25 mM. Fc remained constant at 72 and 120 contractions.min(-1) at 25 and 40 degrees C, respectively, and declined thereafter. The post-rest potentiation was not influenced by ryanodine, indicating that the sarcoplasmic reticulum is not important to the excitation-contraction coupling.     

Short-term storage of oocytes from the neotropical teleost fish Prochilodus marggravii

The loss of oocyte viability after ovulation is one of the limiting factors in controlled reproduction of several fish species. Experiments were performed with 15 feral Prochilodus marggravii female fish induced to spawn with crude carp pituitary extract to evaluate the viability of oocytes retained within the ovarian cavity (in situ storage) and outside of the ovarian cavity (ex situ storage). Because fertility rates rapidly declined after ovulation, simultaneously with an increase in the number of deformed larvae, P. marggravii oocytes could only be successfully stored for 1 h ex situ at room temperature ( approximately 26 degrees C). There was a highly negative correlation (r = -0.82) between fertilization and deformed larvae during in situ storage at approximately 26 degrees C. Ex situ cooling (18 degrees C) caused a drastic reduction in fertilization rates as compared with storage at approximately 26 degrees C. Oocyte structure was preserved during 2 h storage and the cortical reaction was induced before spawning. Since the micropylar apparatus remained open, it was not the primary cause for the loss of oocyte fertility. The cytoskeleton of the oocyte appeared to be affected since ooplasmic segregation was altered after 2 h storage.     

Enameloid in the teleost fish Lophius

Summary The ultrastructural and chemical differences between mature enameloid and dentine in the teleost fish Lophius were demonstrated using transmission (TEM) and scanning (SEM) electron microscopy, X-ray diffraction analysis, electron diffraction analysis and electron microprobe analysis. An enameloid cap (about 50 m thick) and a thin layer (1 m thick) of collar enameloid were revealed in the scanning (SEM) and transmission (TEM) electron microscopes. Collar enameloid, structurally different from dentine both in SEM and TEM, proved to be less mineralized than the cap as revealed by electron microprobe analysis. The enameloid cap was seen to differ from dentine by (i) its electron density in TEM; (ii) the structural arrangement perpendicular to the underlying dentine, observed in SEM and TEM and corroborated by electron diffraction data, which showed a preferred orientation of enameloid crystallites; (iii) its higher F content decreasing gradually from the apex of the tooth to the junction with dentine and falling suddenly below this junction; and (iv) its lower Mg content. Establishment of a classification of enameloids is proposed, similar to that for dentines, in which selachian enameloid would be a reference standard.

---

Résumé La microscopie électronique à transmission, la microscopie électronique à balayage, la diffraction X, la diffraction électronique et l'analyse par microsonde ont prouvé l'existence d'un émailoïde, ultrastructuralement et chimiquement différent de la dentine, chez la baudroie. La microscopie électronique à balayage et la microscopie électronique à transmission ont mis en évidence un chapeau d'émailoïde, d'environ 50 m d'épaisseur, et une couche fine (1 m) d'émailoïde du collet. L'émailoïde du collet, dont l'ultrastructure différente de celle de la dentine a été mise en évidence en microscopie électronique à transmission et en microscopie électronique à balayage, apparaît, après analyse par microsonde, moins minéralisé que le chapeau. Le chapeau d'émailoïde diffère de la dentine par (i) sa densité aux électrons en microscopie électronique à transmission; (ii) sa structure perpendiculaire à celle de la dentine sous-jacente, visible en microscopie électronique à transmission et en microscopie électronique à balayage et confirmée par la diffraction électronique, qui montre une orientation préférentielle des cristaux; (iii) son contenu en fluor plus élevé, qui diminue progressivement de l'apex à la jonction avec la dentine et chute brutalement au-dessous de cette jonction; (iv) sa teneur plus basse en magnésium. Les auteurs proposent que soit établie une classification des émailoïdes analogue à celle des dentines et dans laquelle l'émailoïde de Sélaciens constituerait un standard de référence.

     

Structure of the Tetrahymena thermophila telomerase RNA helix II template boundary element

Telomere addition by telomerase requires an internal templating sequence located in the RNA subunit of telomerase. The correct boundary definition of this template sequence is essential for the proper addition of the nucleotide repeats. Incorporation of incorrect telomeric repeats onto the ends of chromosomes has been shown to induce chromosomal instability in ciliate, yeast and human cells. A 5′ template boundary defining element (TBE) has been identified in human, yeast and ciliate telomerase RNAs. Here, we report the solution structure of the TBE element (helix II) from Tetrahymena thermophila telomerase RNA. Our results indicate that helix II and its capping pentaloop form a well-defined structure including unpaired, stacked adenine nucleotides in the stem and an unusual syn adenine nucleotide in the loop. A comparison of the T.thermophila helix II pentaloop with a pentaloop of the same sequence found in the 23S rRNA of the Haloarcula marismortui ribosome suggests possible RNA and/or protein interactions for the helix II loop within the Tetrahymena telomerase holoenzyme.