Browsing by Subject "Genetic regulation"

Now showing 1 - 8 of 8

Directed evolution of T7 RNA polymerase variants using an 'autogene'
(2004-08) Chelliserrykattil, Jijumon Pavithran, 1974-; Ellington, Andrew D.
Natural enzymes, when used in biotechnological applications are often found not well suited for the tasks. Enzyme properties can now be improved by rational design or by directed evolution to produce useful biocatalysts. The work described in this thesis is mainly focused towards developing a directed evolution method to evolve RNA polymerases with novel properties, such as the ability to use modified nucleotides as substrates. A variety of modified nucleotides can be imagined that can impart unique characteristics to RNA molecules into which they are incorporated. Modified nucleotides improve the functionality of RNA, but more importantly, they increase the stability of RNA towards nucleases thus lending the RNA amenable to various biotechnological applications. Modified RNA transcripts will also find applications in the in vitro selection of aptamers and ribozymes. We have developed a directed evolution method for the isolation of RNA polymerase variants with altered promoter specificities and novel substrate specificities vii using a construct called an “autogene”. The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNA polymerase) is being used for the directed evolution studies. In short, a library of T7 RNA polymerase variants was made by randomizing the gene of T7 RNA polymerase at amino acid positions that are important for a desired activity (for e.g.: altered promoter recognition). This gene was then cloned downstream of a T7 promoter, generating a so-called autogene library. Following transformation to E.coli, those polymerase variants that best recognized their adjacent promoter self-amplified both their mRNAs and themselves in vivo. The variant mRNAs extracted from the population as a whole will be roughly represented according to the activities of their corresponding variant polymerases. Following reverse transcription and PCR amplification in vitro, the most abundant polymerase genes were carried into subsequent rounds of selection. The method allows large (103 -106 ) polymerase libraries to be efficiently searched for their promoter recognition ability and fidelity. Autogene selection was subsequently modified with a reporter gene and used to screen polymerase variants that can incorporate modified nucleotides into the RNA backbone. We have successfully evolved a novel T7 RNA polymerase variant that transcribes 2’-O-methyl RNA. Other selections can also be envisioned using the autogene system to discover new polymerases with novel abilities. The polymerases thus evolved were used to construct modified RNA libraries to be used in in vitro selection of modified ribozymes.
Dynamics of adaptive evolution in two experimental viral systems
(2001-12) Holder, Kristina Kichler; Bull, James J.; Hillis, David M., 1958-
Engineered regulation of an RNA ligase ribozyme
(2001-08) Robertson, Michael Paul; Ellington, Andrew D.
Catalytic RNA has been implicated as a critical component of the origin of life on Earth. Compelling arguments have been made for an ‘RNA world’ era during the early evolution of life in which all living systems consisted solely of RNA functioning as both genetic storage media and catalytic agents. Since the discovery of catalytic RNA in the early 1980s, the types of chemical reactions that RNA has been shown to catalyze has steadily increased and with it the feasibility of an all-RNA metabolism. However, a key aspect of modern biocatalysts is their ability to attenuate their level of activity in response to changing environmental conditions. Presumably, a similar regulation strategy would also be necessary to sustain a reasonably complex RNA-based metabolism. We have isolated and designed several RNA ligase ribozymes whose activities are regulated by the presence of a variety of different effectors. An oligonucleotidedependent ligase (L1) was isolated from a randomized pool of RNA using in vitro selection. Other allosterically regulated ribozymes were engineered by replacing a non-essential stem loop of L1 with various small molecule binding RNA aptamers to create aptamer-ribozyme hybrids (aptazymes). Alternatively, we vi have employed a two stage in vitro selection procedure with a partially randomized pool based on the L1 ligase to isolate protein-activated aptazymes that are extremely dependent on their cognate effector for activity. In addition to the regulation of RNA catalysis, we have been investigating ways to increase the proficiency and versatility of ribozymes by augmenting the chemical functional groups of RNA by the incorporation of modified nucleosides. These modified nucleotides can be efficiently transcribed into RNA using T7 RNA polymerase and have been incorporated into random sequence RNA pools for use in in vitro selection experiments. These results demonstrate that ribozyme activity can be regulated in much the same way that protein enzymes are regulated in contemporary biochemistry. This ability of RNA to be controlled by various potential metabolic intermediates contributes an additional layer of sophistication to ribozyme catalysis and increases the plausibility that a complex metabolism based solely on RNA could have once existed.
Exploring the global gene expression programs and regulation in the response of quiescent human fibroblasts to distinct proliferative stimuli
(2005) Gu, Jian; Iyer, Vishwanath R.
Serum treatment of quiescent human dermal fibroblasts induces proliferation coupled with a complex physiological response that is indicative of their normal role in wound-healing. However, it is not known to what extent such complex transcriptional events are specific to a given cell type and signal, and to what extent these changes are innate programmed responses that are activated in a range of related cell types in response to a variety of stimuli. We have profiled the global transcriptional program of human fibroblasts from two distinct tissue sources to four different growth stimuli and identified a striking conservation in their gene expression signatures. However, there were specific differences among different stimuli with regard to signaling pathways that mediate these transcriptional programs. The use of a specific PI3-kinase pathway inhibitor suggested that this pathway is differentially involved in mediating the responses of cells to serum as compared to individual peptide growth factors. By applying siRNA knockdown technique, we demonstrated that putative targets of two important immediate early transcription factors, Myc and SRF, served functions related to cell cycle progression/cell survival and wound healing, indicating that these two transcription factors may serve as master transcription controllers during the transition of fibroblasts from quiescence to proliferation. In addition, different Myc targets were identified either between different cell types (Hela vs. foreskin fibroblasts) or between different cell states (unsynchronized vs. synchronized), while SRF targets included a group of genes only induced at certain time points during cell cycle progression, which was not observed in the Myc data. MicroRNA (miRNA) expression profiling indicated that let7 and other miRNAs with similar expression profiles may be involved in regulating the transcriptional program in response to proliferative signals. Our results indicate that conservation of transcriptional programs and their regulation among different cell types may be much broader than previously appreciated.
From developing protein-protein interaction strategies to identifying gene functions: case studies for transcription factor complexes and ribosome biogenesis genes
(2007-12) Li, Zhihua, doctor of cell and molecular biology; Marcotte, Edward M.
Protein-protein interactions are central to their biological functions in cells. Many approaches have been applied to study protein-protein interactions in a genomic-scale. In an attempt to develop new strategies to study protein-protein interactions, FRET by using ECFP and EYFP as the donor and receptor was evaluated for possible application in protein-protein interaction study in a high-throughput fashion. Due to the intrinsic properties of ECFP and EYFP, FRET-based protein-protein interaction assay is not suitable for large-scale studies. Instead, tandem affinity purification coupled with mass spectrometry approach proved to be a useful strategy to identify protein interacting partners. Several transcription factor complexes in yeast were successfully purified and novel components in the complexes were identified by combining a shotgun mass spectrometry approach and a differential analysis of the mass spectrometry data. In particular, a negative regulator of G1 to S phase transition during cell cycle, Whi5p, was identified to be a component of SBF complex; a regulator of nitrogen metabolism, Gln3p, was identified to be a component of Hap2/3/5 complex that regulates carbon metabolism, suggesting a crosstalk between nitrogen and carbon metabolism. Additionally, one-step purification coupled with shotgun mass spectrometry analysis was applied to simplify and improve the affinity purification approach used for protein-protein interaction studies. In order to map protein complexes in their native state, a sucrose density gradient was used to separate protein complexes in cells. The proteins within each fraction from the sucrose density gradient were analyzed and quantified with mass spectrometry to obtain the protein abundance profiles across the gradient. The known protein complexes were identified by clustering the protein abundance profiles. This method could possibly be improved to become a generic approach to mapping protein complexes. The goal of protein-protein interaction studies is to determine the protein functions. In an effort to identify ribosome biogenesis genes from a yeast gene network reconstructed from diverse large-scale interaction data sets, at least 25 new ribosome biogenesis genes were confirmed by extensive experimental validations, underscoring the value of proteinprotein interaction studies and gene interaction network.
Functional characterization of m-Bop, a transcriptional repressor essential for heart development
(2002) Sims, Robert Joseph; Gottlieb, Paul D.
The Bop gene encodes MYND and SET domain-containing proteins that are expressed in cytotoxic T lymphocytes (t-Bop) and in skeletal and cardiac muscle (m-Bop). MYND and SET domains are found in numerous transcriptional regulators and chromatin remodeling proteins. The MYND domain is believed to function as a protein-protein interaction motif, while the SET domain was recently demonstrated to participate in core histone covalent modification. Previous studies in mice demonstrated that Bop is required for proper cardiac development, specifically the right ventricle. Additionally, targeted inactivation of the Bop gene altered the expression of chamber-specific transcription factors. While much has been learned from prior experimentation, the molecular function of Bop has not been previously described. In these studies, Bop is shown to function as a histone deacetylase-dependent transcriptional repressor. When tethered to the GAL4-DNA binding domain, m-Bop and t-Bop repressed the expression of a luciferase reporter in a dose-dependent fashion. The addition of trichostatin A, a potent inhibitor of histone deacetylase (HDAC) activity, significantly reduced the repression potential of m-Bop. Coimmunoprecipitation experiments identified that m-Bop physically associates with class I and class II HDACs in cell culture. The nuclear receptor co-repressor N-CoR interacted with m-Bop in similar assays. Furthermore, m-Bop displayed the ability to associate with multiple components of the SIN3 repression complex in vivo. While SET domain-dependent histone methyltransferases (HMTases) have been recently identified, m-Bop did not demonstrate HMTase activity in vitro, suggesting that Bop may function as a ‘mute’ HMTase. Physical interaction between m-Bop and skNAC, a reported muscle-specific transcriptional activator, was detected in the yeast two-hybrid system and binding assays in vitro and in vivo. m-Bop and skNAC were induced and localized in a similar manner during skeletal myogenesis in vitro. The muscle-specific and MYND domains of m-Bop were required for skNAC association in mammalian cells. skNAC residues important for m-Bop interaction were located near a carboxy terminal proline-rich region. Computational analysis of Bop identified it as the charter member of a family of MYND and SET domain proteins found in a broad range of species including yeast, plants, worms, flies, mice, and humans.
Functional characterization of smyd1, a methyltransferase essential for heart and skeletal muscle development
(2006) Zhu, Li; Tucker, Philip W.
Post-translational modifications of histone tails have fundamental roles in chromatin structure and function. Among chromatin modifying enzymes, SET domain proteins were identified as histone lysine methyltransferases that play critical roles in the regulation of gene expression. Until recently, the studies of SET domain proteins have mainly concentrated on histones. The discovery of a few nonhistone substrates of SET domain proteins demonstrated that histones are not the sole substrates of this family and that methylation of nonhistone regulatory proteins may play an equally important role in transcription regulation. Among the SET domain proteins, SMYD1a and b, two isoforms that differ by a 13 amino acid insertion in the SET domain due to alternative splicing, were identified as heart and skeletal muscle-specific proteins essential for cardiogenesis and skeletal muscle development. In this thesis, I show that in vitro, both SMYD1a and SMYD1b methylate skNAC, a cardiac and skeletal muscle specific transcriptional activator. In vivo, SMYD1b but not SMYD1a methylates skNAC. The methylation of skNAC requires a direct interaction of SMYD1 and skNAC. The expression of myoglobin, a heart and skeletal muscle-specific hemoprotein that facilitates oxygen transport in cardiomyocytes and skeletal muscle is regulated by both SMYD1 and skNAC. skNAC regulates myoglobin expression by directly binding to the myoglobin promoter, while SMYD1 regulate myoglobin expression indirectly, likely through skNAC methylation. The ability of SMYD1-depleted C2C12 myoblasts to differentiate into myotubes was impaired, indicating SMYD1 plays critical roles in the early stage of myoblastic differentiation. However, skNAC is not required for the early stage of differentiation, as skNAC-depleted myoblasts differentiate into myotubes normally.
Pax6 and Six1/2 orthologs in leech ectodermal patterning
(2008-12) Quigley, Ian Kirk; Shankland, Marty
Clitellate annelids display conserved mechanisms of segmental ectodermal and mesodermal patterning. These tissues are generated by asymmetric divisions of large stem cells called teloblasts, elongating the ectoderm and mesoderm of the embryo. Each teloblast-derived lineage makes highly stereotyped contributions to the leech: the N, O, P, and Q contribute specific neurons, epidermis, and other ectodermal tissues along the ventral-to-dorsal axis of the embryo, respectively. The N and Q ectodermal lineages appear to be specified autonomously, but specification of the O and P lineages depends upon interactions with other, neighboring teloblast lineages. Until quite recently, there have been precious few teloblast lineage-specific markers, and virtually no molecular candidates for genes influencing the proper differentiation of any of these lineages. Here, I explore the possibility that members of the Pax-Six-Eyes absent-Dachshund network are involved in leech ectodermal patterning. I show that the leech Helobdella sp. Austin has two Pax6 paralogs, and demonstrate that Hau-Pax6A is expressed early in a subset of N-derived cells and O-derived cells. Next, I demonstrate that an ortholog of the six gene family, Hau-six1/2a, is expressed in the P lineage. I show through a series of cell ablations that Hau-six1/2a expression is regulated by neighboring teloblasts in a manner consistent with P fate induction, hinting that this transcription factor may be involved in P specification. The identification of these genes is a first step towards dissecting the molecular mechanisms of ectodermal teloblast differentiation in the leech embryo. The evolutionary context of the deployment of these genes is also discussed. In the appendices, I present two projects on the evolution of pigment patterns in Danio rerio and its relatives. In the first, I show that the larval melanin-containing pigment cells of Danio nigrofasciatus are uniquely redeployed into the adult pigment pattern, in contrast to seven related fishes. In the second, I show that variation in yellow pigment cell populations in different danio species may be dependent on variable signaling through the receptor tyrosine kinase fms pathway.