Synthetic molecular machines for active self-assembly : prototype algorithms, designs, and experimental study

Computer science and electrical engineering have been the great success story of the twentieth century. The neat modularity and mapping of a language onto circuits has led to robots on Mars, desktop computers and smartphones. But these devices are not yet able to do some of the things that life takes for granted: repair a scratch, reproduce, regenerate, or grow exponentially fast–all while remaining functional.

This thesis explores and develops algorithms, molecular implementations, and theoretical proofs in the context of “active self-assembly” of molecular systems. The long-term vision of active self-assembly is the theoretical and physical implementation of materials that are composed of reconfigurable units with the programmability and adaptability of biology’s numerous molecular machines. En route to this goal, we must first find a way to overcome the memory limitations of molecular systems, and to discover the limits of complexity that can be achieved with individual molecules.

One of the main thrusts in molecular programming is to use computer science as a tool for figuring out what can be achieved. While molecular systems that are Turing-complete have been demonstrated [Winfree, 1996], these systems still cannot achieve some of the feats biology has achieved.

One might think that because a system is Turing-complete, capable of computing “anything,” that it can do any arbitrary task. But while it can simulate any digital computational problem, there are many behaviors that are not “computations” in a classical sense, and cannot be directly implemented. Examples include exponential growth and molecular motion relative to a surface.

Passive self-assembly systems cannot implement these behaviors because (a) molecular motion relative to a surface requires a source of fuel that is external to the system, and (b) passive systems are too slow to assemble exponentially-fast-growing structures. We call these behaviors “energetically incomplete” programmable behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time.

As we will demonstrate and prove, a sufficiently expressive implementation of an “active” molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the the problem, so the system is not “energetically incomplete.” But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive.

Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly.

We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self- assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics.

We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic linear polymer that grows exponentially fast via insertion. We show that monomer molecules are converted into the polymer in logarithmic time via spectrofluorimetry and gel electrophoresis experiments. We also demonstrate the division of these polymers via the addition of a single DNA complex that competes with the insertion mechanism. This shows the growth of a population of polymers in logarithmic time. We characterize the DNA insertion mechanism that we utilize in Chapter 4. We experimentally demonstrate that we can control the kinetics of this re- action over at least seven orders of magnitude, by programming the sequences of DNA that initiate the reaction.

In addition, we review co-authored work on programming molecular robots using prescriptive landscapes of DNA origami; this was the first microscopic demonstration of programming a molec- ular robot to walk on a 2-dimensional surface. We developed a snapshot method for imaging these random walking molecular robots and a CAPTCHA-like analysis method for difficult-to-interpret imaging data.

Tissue engineering active biological machines : bio-inspired design, directed self-assembly, and characterization of muscular pumps simulating the embryonic heart

Biological machines are active devices that are comprised of cells and other biological components. These functional devices are best suited for physiological environments that support cellular function and survival. Biological machines have the potential to revolutionize the engineering of biomedical devices intended for implantation, where the human body can provide the required physiological environment. For engineering such cell-based machines, bio-inspired design can serve as a guiding platform as it provides functionally proven designs that are attainable by living cells. In the present work, a systematic approach was used to tissue engineer one such machine by exclusively using biological building blocks and by employing a bio-inspired design. Valveless impedance pumps were constructed based on the working principles of the embryonic vertebrate heart and by using cells and tissue derived from rats. The function of these tissue-engineered muscular pumps was characterized by exploring their spatiotemporal and flow behavior in order to better understand the capabilities and limitations of cells when used as the engines of biological machines.

Effects of particle size ratio on single particle motion in colloidal dispersions

The motion of a single Brownian particle of arbitrary size through a dilute colloidal dispersion of neutrally buoyant bath spheres of another characteristic size in a Newtonian solvent is examined in two contexts. First, the particle in question, the probe particle, is subject to a constant applied external force drawing it through the suspension as a simple model for active and nonlinear microrheology. The strength of the applied external force, normalized by the restoring forces of Brownian motion, is the Péclet number, Pe. This dimensionless quantity describes how strongly the probe is upsetting the equilibrium distribution of the bath particles. The mean motion and fluctuations in the probe position are related to interpreted quantities of an effective viscosity of the suspension. These interpreted quantities are calculated to first order in the volume fraction of bath particles and are intimately tied to the spatial distribution, or microstructure, of bath particles relative to the probe. For weak Pe, the disturbance to the equilibrium microstructure is dipolar in nature, with accumulation and depletion regions on the front and rear faces of the probe, respectively. With increasing applied force, the accumulation region compresses to form a thin boundary layer whose thickness scales with the inverse of Pe. The depletion region lengthens to form a trailing wake. The magnitude of the microstructural disturbance is found to grow with increasing bath particle size -- small bath particles in the solvent resemble a continuum with effective microviscosity given by Einstein's viscosity correction for a dilute dispersion of spheres. Large bath particles readily advect toward the minimum approach distance possible between the probe and bath particle, and the probe and bath particle pair rotating as a doublet is the primary mechanism by which the probe particle is able to move past; this is a process that slows the motion of the probe by a factor of the size ratio. The intrinsic microviscosity is found to force thin at low Péclet number due to decreasing contributions from Brownian motion, and force thicken at high Péclet number due to the increasing influence of the configuration-averaged reduction in the probe's hydrodynamic self mobility. Nonmonotonicity at finite sizes is evident in the limiting high-Pe intrinsic microviscosity plateau as a function of bath-to-probe particle size ratio. The intrinsic microviscosity is found to grow with the size ratio for very small probes even at large-but-finite Péclet numbers. However, even a small repulsive interparticle potential, that excludes lubrication interactions, can reduce this intrinsic microviscosity back to an order one quantity. The results of this active microrheology study are compared to previous theoretical studies of falling-ball and towed-ball rheometry and sedimentation and diffusion in polydisperse suspensions, and the singular limit of full hydrodynamic interactions is noted.

Second, the probe particle in question is no longer subject to a constant applied external force. Rather, the particle is considered to be a catalytically-active motor, consuming the bath reactant particles on its reactive face while passively colliding with reactant particles on its inert face. By creating an asymmetric distribution of reactant about its surface, the motor is able to diffusiophoretically propel itself with some mean velocity. The effects of finite size of the solute are examined on the leading order diffusive microstructure of reactant about the motor. Brownian and interparticle contributions to the motor velocity are computed for several interparticle interaction potential lengths and finite reactant-to-motor particle size ratios, with the dimensionless motor velocity increasing with decreasing motor size. A discussion on Brownian rotation frames the context in which these results could be applicable, and future directions are proposed which properly incorporate reactant advection at high motor velocities.

Spinal cord injury therapy through active learning

Therapy employing epidural electrostimulation holds great potential for improving therapy for patients with spinal cord injury (SCI) (Harkema et al., 2011). Further promising results from combined therapies using electrostimulation have also been recently obtained (e.g., van den Brand et al., 2012). The devices being developed to deliver the stimulation are highly flexible, capable of delivering any individual stimulus among a combinatorially large set of stimuli (Gad et al., 2013). While this extreme flexibility is very useful for ensuring that the device can deliver an appropriate stimulus, the challenge of choosing good stimuli is quite substantial, even for expert human experimenters. To develop a fully implantable, autonomous device which can provide useful therapy, it is necessary to design an algorithmic method for choosing the stimulus parameters. Such a method can be used in a clinical setting, by caregivers who are not experts in the neurostimulator's use, and to allow the system to adapt autonomously between visits to the clinic. To create such an algorithm, this dissertation pursues the general class of active learning algorithms that includes Gaussian Process Upper Confidence Bound (GP-UCB, Srinivas et al., 2010), developing the Gaussian Process Batch Upper Confidence Bound (GP-BUCB, Desautels et al., 2012) and Gaussian Process Adaptive Upper Confidence Bound (GP-AUCB) algorithms. This dissertation develops new theoretical bounds for the performance of these and similar algorithms, empirically assesses these algorithms against a number of competitors in simulation, and applies a variant of the GP-BUCB algorithm in closed-loop to control SCI therapy via epidural electrostimulation in four live rats. The algorithm was tasked with maximizing the amplitude of evoked potentials in the rats' left tibialis anterior muscle. These experiments show that the algorithm is capable of directing these experiments sensibly, finding effective stimuli in all four animals. Further, in direct competition with an expert human experimenter, the algorithm produced superior performance in terms of average reward and comparable or superior performance in terms of maximum reward. These results indicate that variants of GP-BUCB may be suitable for autonomously directing SCI therapy.

Active interaction control for civil structures

This thesis presents a civil engineering approach to active control for civil structures. The proposed control technique, termed Active Interaction Control (AIC), utilizes dynamic interactions between different structures, or components of the same structure, to reduce the resonance response of the controlled or primary structure under earthquake excitations. The primary control objective of AIC is to minimize the maximum story drift of the primary structure. This is accomplished by timing the controlled interactions so as to withdraw the maximum possible vibrational energy from the primary structure to an auxiliary structure, where the energy is stored and eventually dissipated as the external excitation decreases. One of the important advantages of AIC over most conventional active control approaches is the very low external power required.

In this thesis, the AIC concept is introduced and a new AIC algorithm, termed Optimal Connection Strategy (OCS) algorithm, is proposed. The efficiency of the OCS algorithm is demonstrated and compared with two previously existing AIC algorithms, the Active Interface Damping (AID) and Active Variable Stiffness (AVS) algorithms, through idealized examples and numerical simulations of Single- and Multi-Degree-of Freedom systems under earthquake excitations. It is found that the OCS algorithm is capable of significantly reducing the story drift response of the primary structure. The effects of the mass, damping, and stiffness of the auxiliary structure on the system performance are investigated in parametric studies. Practical issues such as the sampling interval and time delay are also examined. A simple but effective predictive time delay compensation scheme is developed.

The application of trimethylsilyl diazomethane to the synthesis of optically active pyrazolines and related studies

The 1,3-dipolar cycloadditions of trimethylsilyl diazomethane with camphorsultam-derived acrylates are reported as a means for the efficient synthesis of optically active pyrazolines. Trimethylsilyl diazomethane is a safe, commercially available diazoalkane which provides Δ¹-pyrazolines 1n good yield and diastereoselectivity when camphorsultam-derived acrylates are used as the reaction dipolarophiles . These initial cycloadducts are subsequently converted to stable, characterizable Δ²-pyrazolines upon desilylation.

A manifold of reactions that can be applied to these Δ²-pyrazolines has been developed which includes pyrazoline reduction, N-N bond reduction, addition to the pyrazoline C=N by mild carbon nucleophiles, and both solvolytic and reductive chiral auxiliary removal. Additionally, it has been demonstrated that the pyrazoline reduction products can take part in peptide coupling reactions that allow for the pyrazolidines to serve as proline-like molecules. The development of this methodology is a general solution to the problem of highly substituted, functionalized pyrazoline synthesis. Importantly, the pyrazolines thus provided have been demonstrated to be amenable to reactions that add to their value as synthetic intermediates.

Transition metal clusters supported by multinucleating ligand frameworks as models of biological active sites

This dissertation describes efforts to model biological active sites with small molecule clusters. The approach used took advantage of a multinucleating ligand to control the structure and nuclearity of the product complexes, allowing the study of many different homo- and heterometallic clusters. Chapter 2 describes the synthesis of the multinucleating hexapyridyl trialkoxy ligand used throughout this thesis and the synthesis of trinuclear first row transition metal complexes supported by this framework, with an emphasis on tricopper systems as models of biological multicopper oxidases. The magnetic susceptibility of these complexes were studied, and a linear relation was found between the Cu-O(alkoxide)-Cu angles and the antiferromagnetic coupling between copper centers. The triiron(II) and trizinc(II) complexes of the ligand were also isolated and structurally characterized.

Chapter 3 describes the synthesis of a series of heterometallic tetranuclear manganese dioxido complexes with various incorporated apical redox-inactive metal cations (M = Na⁺, Ca²⁺, Sr²⁺, Zn²⁺, Y³⁺). Chapter 4 presents the synthesis of heterometallic trimanganese(IV) tetraoxido complexes structurally related to the CaMn₃ subsite of the oxygen-evolving complex (OEC) of Photosystem II. The reduction potentials of these complexes were studied, and it was found that each isostructural series displays a linear correlation between the reduction potentials and the Lewis acidities of the incorporated redox-inactive metals. The slopes of the plotted lines for both the dioxido and tetraoxido clusters are the same, suggesting a more general relationship between the electrochemical potentials of heterometallic manganese oxido clusters and their “spectator” cations. Additionally, these studies suggest that Ca²⁺ plays a role in modulating the redox potential of the OEC for water oxidation.

Chapter 5 presents studies of the effects of the redox-inactive metals on the reactivities of the heterometallic manganese complexes discussed in Chapters 3 and 4. Oxygen atom transfer from the clusters to phosphines is studied; although the reactivity is kinetically controlled in the tetraoxido clusters, the dioxido clusters with more Lewis acidic metal ions (Y³⁺ vs. Ca²⁺) appear to be more reactive. Investigations of hydrogen atom transfer and electron transfer rates are also discussed.

Appendix A describes the synthesis, and metallation reactions of a new dinucleating bis(N-heterocyclic carbene)ligand framework. Dicopper(I) and dicobalt(II) complexes of this ligand were prepared and structurally characterized. A dinickel(I) dichloride complex was synthesized, reduced, and found to activate carbon dioxide. Appendix B describes preliminary efforts to desymmetrize the manganese oxido clusters via functionalization of the basal multinucleating ligand used in the preceding sections of this dissertation. Finally, Appendix C presents some partially characterized side products and unexpected structures that were isolated throughout the course of these studies.

Biophysics of V(D)J recombination and Genome Packaging: In singulo studies on RAG, HMGB1, and TFAM

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.

Suspension mechanics: I. Inertial and non-Newtonian migration of neutrally buoyant rigid spheres in two-dimensional unidirectional flows; II. The effect of viscoelasticity on the creeping motion of a train of neutrally buoyant Newtonian drops through a circular tube

The lateral migration of neutrally buoyant rigid spheres in two-dimensional unidirectional flows was studied theoretically. The cases of both inertia-induced migration in a Newtonian fluid and normal stress-induced migration in a second-order fluid were considered. Analytical results for the lateral velocities were obtained, and the equilibrium positions and trajectories of the spheres compared favorably with the experimental data available in the literature. The effective viscosity was obtained for a dilute suspension of spheres which were simultaneously undergoing inertia-induced migration and translational Brownian motion in a plane Poiseuille flow. The migration of spheres suspended in a second-order fluid inside a screw extruder was also considered.

The creeping motion of neutrally buoyant concentrically located Newtonian drops through a circular tube was studied experimentally for drops which have an undeformed radius comparable to that of the tube. Both a Newtonian and a viscoelastic suspending fluid were used in order to determine the influence of viscoelasticity. The extra pressure drop due to the presence of the suspended drops, the shape and velocity of the drops, and the streamlines of the flow were obtained for various viscosity ratios, total flow rates, and drop sizes. The results were compared with existing theoretical and experimental data.

Multilayer Active Shell Mirrors

This thesis presents a novel active mirror technology based on carbon fiber composites and replication manufacturing processes. Multiple additional layers are implemented into the structure in order to provide the reflective layer, actuation capabilities and electrode routing. The mirror is thin, lightweight, and has large actuation capabilities. These features, along with the associated manufacturing processes, represent a significant change in design compared to traditional optics. Structural redundancy in the form of added material or support structures is replaced by thin, unsupported lightweight substrates with large actuation capabilities.

Several studies motivated by the desire to improve as-manufactured figure quality are performed. Firstly, imperfections in thin CFRP laminates and their effect on post-cure shape errors are studied. Numerical models are developed and compared to experimental measurements on flat laminates. Techniques to mitigate figure errors for thicker laminates are also identified. A method of properly integrating the reflective facesheet onto the front surface of the CFRP substrate is also presented. Finally, the effect of bonding multiple initially flat active plates to the backside of a curved CFRP substrate is studied. Figure deformations along with local surface defects are predicted and characterized experimentally. By understanding the mechanics behind these processes, significant improvements to the overall figure quality have been made.

Studies related to the actuation response of the mirror are also performed. The active properties of two materials are characterized and compared. Optimal active layer thicknesses for thin surface-parallel schemes are determined. Finite element simulations are used to make predictions on shape correction capabilities, demonstrating high correctabiliity and stroke over low-order modes. The effect of actuator saturation is studied and shown to significantly degrade shape correction performance.

The initial figure as well as actuation capabilities of a fully-integrated active mirror prototype are characterized experimentally using a Projected Hartmann test. A description of the test apparatus is presented along with two verification measurements. The apparatus is shown to accurately capture both high-amplitude low spatial-frequency figure errors as well as those at lower amplitudes but higher spatial frequencies. A closed-loop figure correction is performed, reducing figure errors by 94%.

Heterometallic Complexes as Models of Enzymatic Active Sites

This dissertation describes studies on two multinucleating ligand architectures: the first scaffold was designed to support tricopper complexes, while the second platform was developed to support tri- and tetrametallic clusters.

In Chapter 2, the synthesis of yttrium (and lanthanide) complexes supported by a tripodal ligand framework designed to bind three copper centers in close proximity is described. Tricopper complexes were shown to react with dioxygen in a 1:1 [Cu₃]/O₂ stoichiometry to form intermediates in which the O–O bond was fully cleaved, as characterized via UV-Vis spectroscopy and determination of the reaction stoichiometry. Pre-arrangement of the three Cu centers was pivotal to cooperative O₂ activation, as mono-copper complexes reacted differently with dioxgyen. The reactivity of the observed intermediates was studied with various substrates (reductants, O-atom acceptors, H-atom donors, Brønsted acids) to determine their properties. In Chapter 3, the reactivity of the same yttrium-tricopper complex with nitric oxide was explored. Reductive coupling to form a trans-hyponitrite complex (characterized by X-ray crystallography) was observed via cooperative reactivity by an yttrium and a copper center on two distinct tetrametallic units. The hyponitrite complex was observed to release nitrous oxide upon treatment with a Brønsted acid, supporting its viability as an intermediate in nitric oxide reduction to nitrous oxide.

In Chapter 4, a different multinucleating ligand scaffold was employed to synthesize heterometallic triiron clusters containing one oxide and one hydroxide bridges. The effects of the redox-inactive, Lewis acidic heterometals on redox potential was studied by cyclic voltammetry, unveiling a linear correlation between redox potential and heterometal Lewis acidity. Further studies on these complexes showed that the Lewis acidity of the redox-inactive metals also affected the oxygen-atom transfer reactivity of these clusters. Comparisons of this reactivity with manganese systems, collaborative efforts to reassign the structures of related manganese oxo-hydroxo clusters, and synthetic attempts to access related dioxo clusters are also described.

In Appendix A, ongoing efforts to synthesize new clusters supported by the same multinucleating ligand platform are described. Studies of novel approaches towards ligand exchange in tetrametallic clusters and incorporation of new supporting and bridging ligand motifs in trinuclear complexes are presented.

Time-dependent monoenergetic neutron transport in two adjacent semi-infinite media

An exact solution to the monoenergetic Boltzmann equation is obtained for the case of a plane isotropic burst of neutrons introduced at the interface separating two adjacent, dissimilar, semi-infinite media. The method of solution used is to remove the time dependence by a Laplace transformation, solve the transformed equation by the normal mode expansion method, and then invert to recover the time dependence.

The general result is expressed as a sum of definite, multiple integrals, one of which contains the uncollided wave of neutrons originating at the source plane. It is possible to obtain a simplified form for the solution at the interface, and certain numerical calculations are made there.

The interface flux in two adjacent moderators is calculated and plotted as a function of time for several moderator materials. For each case it is found that the flux decay curve has an asymptotic slope given accurately by diffusion theory. Furthermore, the interface current is observed to change directions when the scattering and absorption cross sections of the two moderator materials are related in a certain manner. More specifically, the reflection process in two adjacent moderators appears to depend initially on the scattering properties and for long times on the absorption properties of the media.

This analysis contains both the single infinite and semi-infinite medium problems as special cases. The results in these two special cases provide a check on the accuracy of the general solution since they agree with solutions of these problems obtained by separate analyses.

Mechanics of sediment transport and bedrock erosion in steep landscapes

Erosion is concentrated in steep landscapes such that, despite accounting for only a small fraction of Earth’s total surface area, these areas regulate the flux of sediment to downstream basins, and their rugged morphology records transient changes (or lack thereof) in geologic and climatic forcing. Steep landscapes are geomorphically active; large sediment fluxes and rapid landscape evolution rates can create or destroy habitat for humans and wildlife alike, and landslides, debris flows, and floods common in mountainous areas represent a persistent natural and structural hazard. Despite the central role that steep landscapes play in the geosciences and in landscape management, the processes controlling their evolution have been poorly studied compared to lower-gradient areas. This thesis focuses on the basic mechanics of sediment transport and bedrock incision in steep landscapes, as these are the fundamental processes which set the pace and style of landscape evolution. Chapter 1 examines the spatial distribution of slow-moving landslides; these landslides can dominate sediment fluxes to river networks, but the controls on their occurrence are poorly understood. Using a case-study along the San Andreas Fault, California, I show that slow-moving landslides preferentially occur near the fault, suggesting a rock-strength control on landslide distribution. Chapter 2 provides the first field-measurements of incipient sediment motion in streams steeper than 14% and shows a large influence of slope-dependent flow hydraulics and grain-scale roughness on particle motion. Chapter 3 presents experimental evidence for bedrock erosion by suspended sediment, suggesting that, in contrast to prevailing theoretical predictions, suspension-regime transport in steep streams can be the dominant erosion agent. Steep streams are often characterized by the presence of waterfalls and bedrock steps which can have locally high rates of erosion; Chapters 4 and 5 present newly developed, experimentally validated theory on sediment transport through and bedrock erosion in waterfall plunge pools. Finally, Chapter 6 explores the formation of a bedrock slot canyon where interactions between sediment transport and bedrock incision lead to the formation of upstream-propagating bedrock step-pools and waterfalls.

Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.