Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes

Background The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT) ortholog, and provide a comparison with genes of other vertebrates. Results The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in rayfinned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males. Conclusions OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.Radmila Hrdličková, Jiří Nehyba, Shu Ly Lim, Frank Grützner, Henry R Bose J

Yeast telomerase is capable of limited repeat addition processivity

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Telomerase-mediated sequence addition is dictated by a short ‘template’ region of the RNA component. Despite the short template segment, telomerases from many organisms have been shown to mediate the synthesis of long extension products. This synthesis presumably depends on two types of translocation events: simultaneous translocation of the RNA–DNA duplex relative to the active site after each nucleotide incorporation (type I or nucleotide addition processivity), and translocation of the RNA relative to the DNA product after each round of repeat synthesis (type II or repeat addition processivity). In contrast, telomerases from yeasts have been shown to synthesize mostly short products, implying a defect in one or both types of translocation. In this report, we analyzed the processivity of yeast telomerase in vitro, and identified two position-specific elongation barriers within the 5′ region of the RNA template that can account for the synthesis of incomplete first round products. These barriers respond differently to variations in nucleotide concentration, primer sequence and mutations in the catalytic protein subunit, consistent with their having distinct mechanistic bases. In addition, by using optimal primers and high concentrations of dGTP, we were able to detect significant type II translocation by the yeast enzyme. Thus, the difference between the elongation property of yeast and other telomerases appears to be quantitative rather than qualitative. Our results suggest that yeast may be a useful system for investigating the physiologic significance of repeat addition processivity

Modeling of gold nanocrystal assemblies in superlattices and vesicles, and the synthesis of nanocrystals for low-temperature solar cell fabrication

Recently, nanocrystal (NC) research has grown substantially, due to their unique and diverse properties. Their flexibility has led to a wide set of proposed applications, such as contrast agents in biomedical fields, ordered nanostructures for microelectronics/plasmonics, and as a cheaper alternative to chemical vapor deposition (CVD) methods. While great advancements have been made in utilizing NCs, three challenges often arise – difficulty in characterizing complex nano-systems, a lack of theoretical exploration as to how or why nanocrystals assemble, and challenges in exploiting the benefits of nanocrystals while minimizing disadvantageous properties. This dissertation will address each of these issues in specific systems. First, thorough work has been done suggesting that hydrophobic gold nanocrystals can be encapsulated in vesicle bilayers. However, the primary characterization method for this system is Cryo-Transmission Electron Microscopy, which cannot provide adequate resolution and contrast to fully characterize nanostructures. Here, small angle x-ray scattering is explored as a method for revealing detailed information regarding the bilayer structure. vii Next gold nanocrystal superlattices are explored through molecular dynamics (MD) simulations. While many works have shown crystal structure transitions in a variety of systems, a detailed explanation as to why certain crystal structures are preferred has yet to be provided. This work offers detailed MD simulations to reveal details regarding the packing density of various crystal structures and to estimate diffusion coefficients in various packings. Furthermore, the free energy difference between BCC and FCC configurations for a small set of gold nanocrystals is explored by thermodynamic integration. The simulated properties are also compared to a small set of real systems. The second half of this dissertation addresses practical applications of NCs for photovoltaics. Despite manufacturing benefits, it is well known that the small NC grains and insulating capping ligands make it difficult to produce efficient solar cells. Therefore, two approaches to removing these ligands and growing nanocrystal grains are explored. The first approach focuses on further studying CuInSe2 synthesis as a window into grain growth. The second offers an example of a material with favorable properties for grain growth – Cu3BiS3 – and addresses difficulties in producing it.Chemical Engineerin

Self-Assembly and Thermal Stability of Binary Superlattices of Gold and Silicon Nanocrystals

Simple hexagonal (sh) AB₂ binary superlattices (BSLs) of organic ligand-capped silicon (A; 5.40(±9.8%) nm diameter) and gold (B; 1.88(±10.1%) nm diameter) nanocrystals were assembled by evaporation of colloidal dispersions and characterized using transmission electron microscopy (TEM) and grazing incidence small-angle X-ray scattering (GISAXS). When deposited on tilted substrates by slow evaporation, the sh-AB₂ superlattice contracted slightly toward the substrate with centered orthorhombic structure. Heating the BSL to 200 °C in air led to gold coalescence and segregation to the surface of the assembly without disrupting the Si nanocrystal sublattice, thus creating a sh superlattice of Si nanocrystals

Chains, Sheets, and Droplets: Assemblies of Hydrophobic Gold Nanocrystals with Saturated Phosphatidylcholine Lipid and Squalene

Assemblies of saturated 1,2-diacylphosphatidylcholine lipid and hydrophobic dodecanethiol-capped 1.8 nm diameter gold nanocrystals were studied as a function of lipid chain length and the addition of the naturally occurring oil, squalene. The gold nanocrystals formed various lipid-stabilized agglomerates, sometimes fusing with lipid vesicle bilayers. The nanocrystal assembly structure depended on the hydrocarbon chain length of the lipid fatty acids. The lipid with the shortest fatty acid length studied, dilauroylphosphatidylcholine, created extended chains of gold nanocrystals. The lipid with slightly longer fatty acid chains created planar sheets of nanocrystals. Further increases of the fatty acid chain length led to spherical agglomerates. The inclusion of squalene led to lipid- and nanocrystal-coated oil droplets

Chloroform-Enhanced Incorporation of Hydrophobic Gold Nanocrystals into Dioleoylphosphatidylcholine (DOPC) Vesicle Membranes

Vesicles of dioleoylphosphatidylcholine (DOPC) formed by extrusion (liposomes) with hydrophobic alkanethiol-capped Au nanocrystals were studied. Dodecanethiol-capped 1.8-nm-diameter Au nanocrystals accumulate in the lipid bilayer, but only when dried lipid–nanocrystal films were annealed with chloroform prior to hydration. Without chloroform annealing, the Au nanocrystals phase separate from DOPC and do not load into the liposomes. Au nanocrystals with slightly longer capping ligands of hexadecanethiol or with a larger diameter of 4.1 nm disrupted vesicle formation and created lipid assemblies with many internal lamellar attachments

The Role of Ligand Packing Frustration in Body-Centered Cubic (bcc) Superlattices of Colloidal Nanocrystals

This paper addresses the assembly of body centered-cubic (bcc) superlattices of organic ligand-coated nanocrystals. First, examples of bcc superlattices of dodecanethiol-capped Au nanocrystals and oleic acid-capped PbS and PbSe nanocrystals are presented and examined by transmission electron microscopy (TEM) and grazing incidence small-angle X-ray scattering (GISAXS). These superlattices tend to orient on their densest (110) superlattice planes and exhibit a significant amount of {112} twinning. The same nanocrystals deposit as monolayers with hexagonal packing, and these thin films can coexist with thicker bcc superlattice layers, even though there is no hexagonal plane in a bcc lattice. Both the preference of bcc in bulk films over the denser face-centered cubic (fcc) superlattice structure and the transition to hexagonal monolayers can be rationalized in terms of packing frustration of the ligands. A model is presented to calculate the difference in entropy associated with capping ligand packing frustration in bcc and fcc superlattices