Matching Items (17)
Description
Telomerase is a unique reverse transcriptase that has evolved specifically to extend the single stranded DNA at the 3' ends of chromosomes. To achieve this, telomerase uses a small section of its integral RNA subunit (TR) to reiteratively copy a short, canonically 6-nt, sequence repeatedly in a processive manner using a complex and currently poorly understood mechanism of template translocation to stop nucleotide addition, regenerate its template, and then synthesize a new repeat. In this study, several novel interactions between the telomerase protein and RNA components along with the DNA substrate are identified and characterized which come together to allow active telomerase repeat addition. First, this study shows that the sequence of the RNA/DNA duplex holds a unique, single nucleotide signal which pauses DNA synthesis at the end of the canonical template sequence. Further characterization of this sequence dependent pause signal reveals that the template sequence alone can produce telomerase products with the characteristic 6-nt pattern, but also works cooperatively with another RNA structural element for proper template boundary definition. Finally, mutational analysis is used on several regions of the protein and RNA components of telomerase to identify crucial determinates of telomerase assembly and processive repeat synthesis. Together, these results shed new light on how telomerase coordinates its complex catalytic cycle.
ContributorsBrown, Andrew F (Author) / Chen, Julian J. L. (Thesis advisor) / Jones, Anne (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Description
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
Description
Telomerase is a specialized enzyme that adds telomeric DNA repeats to the chromosome ends to counterbalance the progressive telomere shortening over cell divisions. It has two essential core components, a catalytic telomerase reverse transcriptase protein (TERT), and a telomerase RNA (TR). TERT synthesizes telomeric DNA by reverse transcribing a short template sequence in TR. Unlike TERT, TR is extremely divergent in size, sequence and structure and has only been identified in three evolutionarily distant groups. The lack of knowledge on TR from important model organisms has been a roadblock for vigorous studies on telomerase regulation. To address this issue, a novel in vitro system combining deep-sequencing and bioinformatics search was developed to discover TR from new phylogenetic groups. The system has been validated by the successful identification of TR from echinoderm purple sea urchin Strongylocentrotus purpuratus. The sea urchin TR (spTR) is the first invertebrate TR that has been identified and can serve as a model for understanding how the vertebrate TR evolved with vertebrate-specific traits. By using phylogenetic comparative analysis, the secondary structure of spTR was determined. The spTR secondary structure reveals unique sea urchin specific structure elements as well as homologous structural features shared by TR from other organisms. This study enhanced the understanding of telomerase mechanism and the evolution of telomerase RNP. The system that was used to identity telomerase RNA can be employed for the discovery of other TR as well as the discovery of novel RNA from other RNP complex.
ContributorsLi, Yang (Author) / Chen, Julian Jl (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.
ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Telomerase is a reverse transcriptase that is responsible for the addition of telomeric repeats on to the ends of eukaryotic chromosomes. The purple sea urchin, Strongylocentrotus purpuratus, telomerase enzyme is unique in that its telomerase RNA does not contain the ancestrally conserved CR4/5 domain and instead contains the functionally equivalent eCR4/5 domain. Binding between the purple sea urchin TRBD and eCR4/5 domain is currently poorly understood due to eCR4/5's unique structure. In this work the telomerase RNA binding domain, TRBD, of the purple sea urchin telomerase reverse transcriptase, TERT, was fused to maltose binding protein (MBP) using several different short amino acid linkers and purified via amylose column purification. Short amino acid linkers were cloned into the MBP sea urchin TRBD constructs to facilitate better crystallization of the fusion protein. Future work of this project includes testing telomerase RNA binding affinity to the TRBD constructs and determining the crystal structure of the sea urchin TRBD with bound eCR4/5. Elucidating how eCR4/5 binds to the sea urchin TRBD will provide insights into the evolutionary relationship between eCR4/5 and the pseudoknot/template domain of sea urchin telomerase RNA.
ContributorsKing, Robert (Author) / Chen, Julian (Thesis director) / Li, Yang (Committee member) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Unmanned aerial vehicles have received increased attention in the last decade due to their versatility, as well as the availability of inexpensive sensors (e.g. GPS, IMU) for their navigation and control. Multirotor vehicles, specifically quadrotors, have formed a fast growing field in robotics, with the range of applications spanning from surveil- lance and reconnaissance to agriculture and large area mapping. Although in most applications single quadrotors are used, there is an increasing interest in architectures controlling multiple quadrotors executing a collaborative task. This thesis introduces a new concept of control involving more than one quadrotors, according to which two quadrotors can be physically coupled in mid-flight. This concept equips the quadro- tors with new capabilities, e.g. increased payload or pursuit and capturing of other quadrotors. A comprehensive simulation of the approach is built to simulate coupled quadrotors. The dynamics and modeling of the coupled system is presented together with a discussion regarding the coupling mechanism, impact modeling and additional considerations that have been investigated. Simulation results are presented for cases of static coupling as well as enemy quadrotor pursuit and capture, together with an analysis of control methodology and gain tuning. Practical implementations are introduced as results show the feasibility of this design.
ContributorsLarsson, Daniel (Author) / Artemiadis, Panagiotis (Thesis advisor) / Marvi, Hamidreza (Committee member) / Berman, Spring (Committee member) / Arizona State University (Publisher)
Created2016
Description
Telomerase is a special reverse transcriptase that extends the linear chromosome termini in eukaryotes. Telomerase is also a unique ribonucleoprotein complex which is composed of the protein component called Telomerase Reverse Transcriptase (TERT) and a telomerase RNA component (TR). The enzyme from most vertebrate species is able to utilize a short template sequence within TR to synthesize a long stretch of telomeric DNA, an ability termed "repeat addition processivity". By using human telomerase reconstituted both in vitro (Rabbit Reticulocyte Lysate) and in vivo (293FT cells), I have demonstrated that a conserved motif in the reverse transcriptase domain of the telomerase protein is crucial for telomerase repeat addition processivity and rate. Furthermore, I have designed a "template-free" telomerase to show that RNA/DNA duplex binding is a critical step for telomere repeat synthesis. In an attempt to expand the understanding of vertebrate telomerase, I have studied RNA-protein interactions of telomerase from teleost fish. The teleost fish telomerase RNA (TR) is by far the smallest vertebrate TR identified, providing a valuable model for structural research.
ContributorsXie, Mingyi (Author) / Chen, Julian J.L. (Thesis advisor) / Yan, Hao (Committee member) / Wachter, Rebekka M. (Committee member) / Arizona State University (Publisher)
Created2010
Description
Telomeres are structures at the ends of DNA strands that get longer in the DNA of sperm cells as males age. That phenomenon is different for most other types of cells, for which telomeres get shorter as organisms age. In 1992, scientists showed that telomere length (TL) in sperm increases with age in contrast to most cell of most other types. Telomeres are the protective caps at the end of DNA strands that preserve chromosomal integrity and contribute to DNA length and stability. In most cells, telomeres shorten with each cell division due to incomplete replication, though the enzyme telomerase functions in some cell lines that undergo repetitive divisions to replenish any lost length and to prevent degradation. Cells, and therefore organisms, with short telomeres are more susceptible to mutations and genetic diseases. While TL increases in a subset of sperm cells and longer telomeres may prevent early disintegration of DNA, it may also prevent natural mechanisms of apoptosis, or cell death, from occurring in abnormal sperm.
ContributorsYang, Joanna (Author) / Bartlett, Zane (Author) / Turriziani Colonna, Federica (Editor) / Arizona State University. School of Life Sciences. Center for Biology and Society. (Publisher) / Arizona Board of Regents (Publisher)
Created2017-02-07
Description
The Hayflick Limit is a concept that helps to explain the
mechanisms behind cellular aging. The concept states that a normal human
cell can only replicate and divide forty to sixty times before it
cannot divide anymore, and will break down by programmed cell death
or apoptosis. The concept of the Hayflick Limit revised Alexis
Carrel's earlier theory, which stated that cells can replicate
themselves infinitely. Leonard Hayflick developed the concept while
at the Wistar Institute in Philadelphia,
Pennsylvania, in 1965. In his 1974 book Intrinsic
Mutagenesis, Frank Macfarlane Burnet named the concept after
Hayflick. The concept of the Hayflick Limit helped scientists study
the effects of cellular aging on human populations from embryonic
development to death, including the discovery of the effects of
shortening repetitive sequences of DNA, called telomeres, on the
ends of chromosomes. Elizabeth Blackburn, Jack Szostak and Carol
Greider received the Nobel Prize in Physiology or Medicine in 2009
for their work on genetic structures related to the Hayflick
Limit.
ContributorsBartlett, Zane (Author) / Turriziani Colonna, Federica (Editor) / Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia. (Publisher) / Arizona Board of Regents (Publisher)
Created2014-11-14