Cell signaling is often mediated by protein-protein interactions, which must be specific, tunable, and transient to allow agile responsiveness to cellular messages. Due to their unique properties, multivalent, intrinsically disordered proteins make ideal candidates to accomplish these vital tasks. A single protein with multiple binding sites may bind numerous partners,...
In biology, function and disease are closely intertwined, requiring the coordination of hundreds of intricate chemical reactions. Interdisciplinary cooperation between chemical and biological approaches is necessary to decipher the chemical links between physiological and pathological conditions. In this arena, the precision tools made possible with chemical biology can manipulate and...
Faithful segregation of genetic material during cell division is essential to all life on earth. In eukaryotes, the mitotic spindle – a bipolar array consisting of cytoskeletal filaments called microtubules – is the cellular machinery responsible for this function. The mitotic spindle requires both exquisite spatiotemporal organization and the generation...
Intrinsically disordered proteins (IDP) are a class of proteins that lack a three-dimensional structure and their prevalence and diverse functions in the cell have only been discovered relatively recently. The intermediate chain (IC) subunit of the microtubule motor protein complex dynein contains an N-terminal disordered region, N-IC, which is central...
The ferritin-like superfamily (FLSF) of proteins is composed of a wide variety of functionally diverse proteins involved in oxygen dependent metal-mediated electron transfer reactions. Their biological importance is exemplified by the fact FLSF proteins are found in almost every organism from all three domains of life. Their functions range from...
Cell motility and invasion are important for development, immunity, wound healing, and tumor cell metastasis. Cells on two dimensional substrates migrate in three steps: protrusion of the front end, translocation of the cell body, and retraction of the rear end. For cells to migrate efficiently, these steps need to be...
CTIP2 is a C2H2 zinc finger transcription factor that plays important yet poorly understood roles in mouse development. CTIP2 is known to be highly expressed in the central nervous system, skin and T lymphocytes during embryogenesis. CTIP2-null mice die perinatally of unknown causes and exhibit defects in multiple organ systems...
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The Class I and Class II histone deacetylase inhibitor TSA partially
reverses transcriptional
Four-stranded DNA junctions (also known as Holliday junctions) are structural intermediates involved in a growing number of biological processes including DNA repair, genetic recombination, and viral integration. Although previous studies have focused on understanding the conformational variability and sequence-dependent formation of Holliday junctions in solution there have been relatively few...
This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to...
Integrase catalyzes insertion of a retroviral genome into the host chromosome. Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner....
Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in cells. In an in vitro replication system involving semiconservative replication from an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human cell extracts in...
The three dimensional structure of both a ligand and its cognate receptor are required for the success of structure-assisted drug design. This thesis reports the crystal structure of hectochlorin, a small, bioactive molecule, and the steps toward determining the crystal structure of an RNA molecule that is an attractive target...
This thesis examines the interconnection between the in vitro and in
vivo phosphorylation of rabbit muscle phosphofructokinase. The first goal
of the project was to show whether a novel site of rabbit muscle
phosphofructokinase that is subject to in vitro phosphorylation, serine 376,
may also become phosphorylated in vivo. Evidence...
The aim of this research was to gain more understanding on how a linker histone (LH), H1⁰, and high mobility group protein, HMG1, interact with nucleosome. To determine the location of linker proteins, three specific positioning sequences were used to reconstitute core nucleosomes and the binding of H1 and HMG1...
Agrobacterium tumefaciens is a gram-negative soil bacterium
that causes crown gall tumors on dicotyledenous plants. The
transferred DNA (T-DNA) portion of the A. tumefaciens tumor-inducing
(Ti) plasmid enters infected plant cells and integrates into
plant nuclear DNA. The T-DNA is accompanied into plant cells by the
VirD2 endonuclease covalently attached...
A novel protocol for the study of protein-nucleic acid interactions is presented and demonstrated
to be feasible. The protocol combines photochemical crosslinking techniques and mass
spectrometric methods into a new strategy for identifying protein domains or amino acid residues
that are in close contact with nucleic acid in protein-nucleic acid...
Understanding the structure of the 30 nm fiber in chromatin is relevant to understanding eukaryotic replication and transcription. The major controversy among the models of the fiber concerns the disposition of the linker DNA, the DNA between adjacent nucleosomes, and the location of the linker histones. To determine if the...
Time-resolved fluorescence spectroscopy was used to monitor the effects of
varying ionic strength on nucleosome core particle structure. Two main methods were
used in these studies. First, the fluorescence anisotropy decay of bound ethidium was
measured and was shown to reflect the rotational tumbling of the core particle through
solution,...
Ribonucleotide reductase is a remarkable enzyme that catalyzes the rate-limiting
step in the synthesis of the 2'-deoxynucleoside triphosphates. The intent of this project
was to characterize the ribonucleotide reductase encoded by the orthopoxvirus,
vaccinia. The first objective was to study the structural and functional features of the
viral small subunit...
Regulation of expression of three genes in the polyhedron
envelope protein (PEP) gene region of the Orgyia pseudotsugata
multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was
examined. These genes include open reading frame (ORF) 1 (encoding
p21), ORF 2 (encoding gp16), and ORF 3 (encoding the polyhedron
envelope protein). The effect of...
Vaccinia virus infected monkey kidney cells had been previously shown
to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA
from mutant virus resistant to hydroxyurea were digested with restriction
endonucleases and were shown to have substoichiometric amounts of the Hind
III F fragment. Additional information from Southern blotting experiments...
Ribosomes are multicomponent macromolecular particles and
are essential for the survival of cells in all organisms. The ribosome's
universal function is to catalyze polypeptide synthesis through
translation of mRNA transcripts. Ribosomes from Escherichia coli,
eubacterial organisms, have a sedimentation coefficient of 70S and are
composed of 30S and 50S ribonucleoprotein...
By using immobilized trypsin, I have been able to
prepare well-defined, stable trypsinized nucleosomes. The
difficulties of lacking of control in the extent of
trypsinization, which were encountered in previous studies
with the use of free trypsin, have been eliminated. The
nucleosomes and oligonucleosomes prepared by immobilized
trypsin are suitable...
Escherichia coli 16S-like ribosomal RNAs from
eubacterial, archaebacterial and eukaryotic organisms have
several universally conserved sequence zones which are
proposed to be single-stranded. A clear function for all
of these regions has not yet been defined. To evaluate the
roles of these sequence zones in protein synthesis,
knowledge of their...
Ribosomes are ribonucleoprotein particles that are essential for
the process of protein synthesis in all living systems. Escherichia
coli, eubacterial organisms, have ribosomes which display a 70S
sedimentation coefficient. They are formed by the association of two
ribonucleoprotein subunits which sediment at 50S and 30S. The 30S
subunit is a...
Boron has been recognized since 1923 as an essential
micronutrient for vascular plants, but its function is
still not known. Many diverse roles have been proposed,
but none of these has been definitively shown. Problems
that have made the study of the function of boron
difficult include, no useful radioisotope,...
The question of how the presence of nucleosomal
packing of DNA modifies carcinogen interaction at
specific sites cannot be answered by studies on whole
chromatin or bulk nucleosomes because of the
heterogeneity of DNA sequences in the particles. This
problem was circumvented by constructing nucleosomes that
are homogenous in DNA-histone...
Ribosomes are intricate macromolecular complexes which are a major
element of the protein biosynthetic machinery in all life forms. In
Escherichia coli they contain about 50 distinct proteins and 3
ribosomal RNAs. The small 30S ribosomal subunit in E. coli
incorporates 21 proteins and a 16S rRNA. The 16S rRNA...
Five forms of chromatin, which represent the in vivo folded forms
of nucleic acid, were isolated and used as the binding substrate for
model intercalating compound, ethidium bromide. For all forms of
chromatin, the affinity, location and structural effects of binding
were examined. On the level of the nucleosome, the...
The association of hyaluronic acid (HA) into a double stranded
structure has been investigated by circular dichroism (CD)
spectroscopy into the vacuum ultraviolet region. The CD of HA changes
dramatically, monitoring a cooperative transition as the dielectric
constant of an aqueous solution is reduced by adding organic solvents.
This structural...
The consistent application of phosphatase inhibitors and a novel final purification step using a connected series of DE-51, DE-52, and DE-53 anion exchange chromatography columns facilitate the preparation of electrophoretically homogeneous sub-pupulations of rabbit muscle phosphofructokinase which differ in their catalytic properties and endogenous covalent phosphate contents. A band of...
Senecio jacobaea (tansy ragwort) is a common weed in the
Pacific Northwest that contains toxic pyrrolizidine alkaloids
(PAs). Jacobine, the major PA in Senecio jacobaea, was incubated
with rat liver microsomes and metabolites were isolated by high
performance liquid chromatography (HPLC) and identified by mass
spectrometric analysis. Metabolites included a...
The ribosome is a central component of the protein
synthetic apparatus. It is a macromolecular complex of
protein and RNA. Although much progress has been made in
understanding the functional role of the proteins in this
particle, little is known of the functional role which
the RNA plays. Naturally occurring...
Three highly purified forms of cytochrome P-450 (P-450a, P-450b, P-450c) from Aroclor 1254-induced rat hepatic microsomes catalyzed the monooxygenation of 1-(2- chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) in the presence of purified NADPH cytochrome P-450 reductase, NADPH and oxygen. Cytochrome P -450- dependent monooxygenation of CCNU gave six alicyclic hydroxylation products...
Certain 2- chioroethyl nitrosoureas have been demonstrated
to be highly specific inactivators of chymotrypsin
in vitro and glutathione reductase, both in vivo and in
isolated rat hepatocytes. The inactivation of glutathione
reductase in isolated hepatocytes resulted in an increased
sensitivity toward an adriamycin induced oxidative
challenge.
Prolonged incubation of 1-(2-chloroethyl)-3-([1-¹⁴...
The structure of the nucleoprotein core of adenovirus has been
investigated. Using a combination of biophysical techniques and
nuclease digestion I have determined that the adenovirus core, as
isolated with pyridine, shares several physical properties with cellular
chromatin. The most notable difference is the lack of a nucleosome
repeat in...
The nucleosome appears to be the basic structural unit of the
eukaryotic chromosome. The usual nucleosome preparation, isolated
from a micrococcal nuclease digest of nuclei, is heterogenous in DNA
length and protein composition with some particles containing spacer
DNA and associated protein. This heterogeneity complicated physical
and reconstitution studies. As...
The interaction of polylysine with DNA may be divided into two
separate phenomena: the primary interaction involved in formation of
a well-defined complex, and secondary interactions leading to aggregation
of these complexes. Most studies on the interaction of polylysine
with DNA have used techniques which measure properties of
the aggregated...