Philip Serwer - US grants

During the morphogenesis of double-stranded DNA bacteriophages, a DNA-free protein capsid (procapsid) is assembled and subsequently packages the bacteriophage DNA. Procapsids consist of a multimolecular outer shell and two classes of internal proteins that assist, by unknown mechanisms, assembly of the outer shell. Previous studies of the structure and assembly of viruses suggest that assembly of outer procapsid shells kinetically controlled by induction of changes in the conformation in unassembled subunits. By this hypothesis, unassembled subunits in a nonassociating conformation switch to a conformation appropriate for entry into the assembly pathway after contact with a partially assembled precursor of the procapsid (conformational switching). However, details of the extent to which conformational switching occurs are not known for any viral capsid. The long-range goal of the research proposed here is to determine the mechanisms by which procapsids assemble from unassembled proteins. Emphasis is to be placed on determining how internal proteins assist assembly of the procapsid's outer shell and on determining the extent to which assembly is controlled by conformational switching. To reach this goal, the specific aims are to accomplish the following, using the related bacteriophages, T7 and T3, as models: (a) increase understanding of the structure of procapsids; electron microscopy, immuno-electron microscopy, protein-protein crosslinking, controlled proteolysis (based on our previous determination of tertiary structure by this procedure) and laser-Raman spectroscopy will be used, (b) detect and isolate multimolecular potential procapsid precursors that are produced during assembly of procapsids "in vivo" and "in vitro"; for detection and isolation, procedures of gel electrophoresis, density gradient electrophoresis and centrifugation, several recently developed in the laboratory of the PI, will be used, (c) characterize the multimolecular particles isolated using the above procedures and also our newly-developed procedures of gel sieving, (d) determine which of the multimolecular particles isolated are in the assembly pathway of the procapsid and determine the temporal sequence of these particles in the pathway; procedures of kinetic labeling-gel electrophoresis-autoradiography will be used whenever possible, (e) continue our development of gel sieving for determining the shape of a multimolecular particle. The information obtained will assist in understanding mechanisms of the assembly of multimolecular complexes in normal and diseased cells. Some of the procedures developed should have use in diagnostic virology.

During the morphogenesis of several animal and bacterial viruses, a DNA-free protein capsid is assembled and this capsid subsequently packages the viral DNA. During packaging, some bacteriophages cut their genomes from a larger (concatemeric) DNA. Our long-range objective is to understand viral DNA packaging at the molecular level. To accomplish this objective, we will use bacteriophages T3, T7 and P22 as model systems and will work toward the accomplishment of the following: a) isolation of capsid-DNA complexes in the DNA packaging pathway in vivo and in vitro: attempts will be made to isolate capsid-DNA complexes that have in the past been too unstable or short-lived to be observed; electrophoresis in density gradients and velocity sedimentation will be used for isolation; variation of isolation buffers, chemical cross-linking of intermediates and genetic perturbation of intermediates will be used. b) Determination of the temporal order of capsid-DNA complexes in the DNA packaging pathway: kinetic labeling experiments will be performed and attempts to have isolated capsid-DNA complexes re-enter the DNA packaging pathway in vitro will be made. c) Determination of the proteins and nucleotide sequences participating in capsid-DNA binding: immunoelectron microscopy, DNA-protein cross-linking and restriction endonuclease cleavage analysis will be used. d) Determination of the source(s) of energy for DNA packaging: a procedure for packaging DNA in vitro at high efficiency in a chemically defined mixture will be developed and will be used to test the effects of possible sources of energy. e) Determination of the arrangement of partially and totally packaged DNA: new procedures for utilizating DNA-reactive reagents will be used. f) Determination of DNA concatemer cutting mechanisms: restriction endonuclease cleavage analysis of the nature of the cut will be made and isolation of the nuclease will be attempted. g) Further characterization of capsids and DNA participating in DNA packaging: agarose gel electrophoresis, isoelectric focusing, protein-protein cross-linking and electron microscopy will be used. h) Further development of our procedures for isolating and characterizing viral assembly intermediates. The data obtained are expected to be useful in developing procedures of antiviral chemotherapy. Procedures in h) are expected to be useful in detecting, isolating and characterizing disease-causing viruses.

Gel electrophoresis has been used to fractionate macromolecules for studies in all fields of biochemistry. However, the sieving of gels during electrophoresis is not understood physically. Sieving can be used to determine the size of spheres and, in some cases, the molecular weight of random coils. Drs. Serwer and Griess propose to: (a) obtain and use data for the testing of previously proposed hypotheses that quantitatively describe the sieving of gels and (b) determine the effect of a particles's shape on its sieving during gel electrophoresis. These objectives are components of a research program for understanding the assembly and purposeful motion of macromolecules, using bacteriophages as model systems. It is anticipated that achieving these objectives will also serve as a foundation for utilization of gel electrophoresis in several areas of biochemistry, microbiology, colloid chemistry and cellular biology.

9421152 Serwer The excluded volume of macromolecular solutes has dramatic effects on the biochemical activities, conformation and assembly of biological macromolecules. Analysis of in vitro excluded volume effects is performed to both understand in vivo excluded volume effects and enhance the in vitro yield of several biochemical processes of practical interest. In previous work by the PI and coworkers, the first quantitative analysis has been performed of the excluded volume of DNA-polymer interaction in mixtures that consist of both double-stranded DNA and electrically neutral polymers. Both biomolecular joining and cyclization of DNA were studied. These studies have revealed the reason for a dependence of excluded volume on the molecular weight of the neutral polymers. They have also revealed excluded volume-induced protection of high molecular weight DNA from hydrodynamic shear-induced breakage. Incompletely explored areas include statistical analysis of the excluded volume induced changes in the conformation of single DNA molecules. Incompletely explored practical applications include use of excluded volume to either prevent the hydrodynamic shear-induced breakage of double-stranded DNA or increase the rate of in vitro biochemical reactions. The specific aims are the following: (1) By use of video light microscopy, statistical analysis will be performed by the dimensions of single DNA molecules after excluded volume-induced condensation. Results will be obtained for several concentrations and types of polymer. Tests will be made of our current theoretical framework for understanding excluded volume effects. (2) By use of both pulsed and constant field quantitative gel electrophoresis, the extent will be determined to which manipulation of excluded volume can be used to both ligate long DNA and prevent the hydrodynamic shear-induced breakage of long DNA. The research proposed contributes to both fundamental understanding of in vivo biochemistry and the practical use of in vitro biochemistry for the purposes of biotechnology. %%% Addition of chemically nonreactive macromolecules (polyethylene glycol's are often used) to solutions of biological macromolecules (such as DNA) has dramatic effects on both the biochemical activity and the conformation of the biological macromolecules. Some of these effects are caused by restriction of the volume available (excluded volume effects). For double-stranded DNA, some excluded volume effects can be visualized by light microscopy of single DNA molecules; these effects include collapse of a random coil of DNA. The goals of our research are the following: (1) By use of light microscopy, statistical analysis will be performed of the dimensions of single DNA molecules after excluded volume induced collapse. (2) By use of gel electrophoresis, the extent will be determines to which manipulation of excluded volume can be used to both promote the joining of DNA molecules and prevent breakage of DNA that occurs when long DNAs are handled by pipeting. Tests will be made of our current theoretical framework for understanding excluded volume effects. The research proposed contributes to both fundamental understanding of the in vivo excluded volume effects of cellular macromolecules and the practical use of in vitro biochemistry for the purposes of biotechnology. ***

By use of a model system that has several advantageous properties, we propose to investigate the mechanisms by which macromolecules cooperate to achieve a biologically evolved purpose. The model system consists of the components required during the packaging of double-stranded DNA in the DNA-free procapsid of bacteriophage T7. The advantageous properties include accessibility to the following procedures that we have developed for this type of investigation: first, analytical procedures for detecting, characterizing and quantifying particles in intermediate states of packaging (DNA packaging intermediates), and second, a high efficiency (more than 20%) in vitro procedure for producing the end product. In past studies, we h ave analyzed several DNA packaging intermediates, including capsids with incompletely packaged DNA. The data yield a model for both the sequence of events at the beginning of packaging and the mechanism for transduction of energy during entry of DNA into a capsid. The specific aims are the following: (a) By use of both ultracentrifugation and our unusually capable, newly-developed techniques of nondenaturing gel electrophoresis, we will both detect and characterize additional T7 DNA packaging intermediates. Our current model for the DNA packaging pathway will be both tested and, if correct, extended. (b) We will test models for the mechanism of energy transduction during in vitro T7 DNA packaging. For this purpose, video light microscopy will be performed of single DNA molecules being packaged. (c) To assist in comparing in vitro to in vivo results, we will investigate the effects on in vitro DNA packaging of nonspecific factors present in vivo, but not yet accurately mimicked in vitro. Thes four include excluded volume, lowered water activity and the presence of a sieving network. (d) To assist in achieving specific aims (a) and (b) we will investigate the effects of in vitro packaging of removing molecules that are present in vivo, but are not necessary for producing the end product. We will develop an in vitro system of purified components that mimics in vivo DNA packaging. The proposed work will answer questions of general significance concerning biochemical pathways biological energy transduction and the relationship of in vitro to in vivo systems. Answering of these questions will open new approaches to antiviral therapies. This proposal is interdisciplinary.

For both the present and the foreseeable future, the nucleotide sequence of DNA strands is determined by the electrophoresis of an ensemble (ladder) of single-stranded DNAs. Thus far, only polyacrylamide gels have had pores small enough to generate the sieving needed for DNA sequencing. The current procedures are, however, limited to the sequencing of 300-500 nucleotides per fractionation. To reduce both the number of samples prepared for electrophoresis and the difficulties in assembling partial sequences, particularly when the sequence is repetitive, lengthening the readable DNA sequencing ladder is needed. Increasing both the efficiency and reproducibility of DNA sequencing gels would also help to accomplish these goals. Thus, ideally, gels developed to lengthen readable DNA sequencing ladders would also be mass-producible. However, polyacrylamide gels are too storage-unstable for mass production. To lengthen DNA sequencing ladders, both pore size-gradient gels and alternative gel matrices are needed. For satisfying all of these needs, we have found that a newly investigated, gel-forming (non polyacrylamide) polymer has the following promising features: pores sufficiently small for DNA sequencing, probable storage stability, and formation of pore size-gradients by procedures adaptable to mass production. One of these procedures is our procedure for using an ion gradient to produce a pore size-gradient. By use of either this or an improved gel-forming compound, our first two specific aims are the following: (1) For the purposes of developing both robust pore size- gradient gels and improved uniform gels, both of which can be mass- produced for DNA sequencing, we will determine the sieving characteristics of gels as a function of conditions of gelation (ionic strength, pH, buffer type, temperature). We will use our previously- developed procedures to determine the gel's effective pore radius from the observed sieving of spheres. (2) By use of the data obtained under the first specific aim, we will develop procedures for producing both pore size-gradient gels and uniform gels that both lengthen electrophoretic DNA sequencing ladders and provide the advantages of mass-production. By use of field inversion, we will attempt to further improve the resolution of DNA sequencing ladders. Our third specific aim is a response to the needs of investigators who are developing capillary electrophoresis for the sequencing of DNA. (3) In response to these needs, we will explore the production of DNA sequencing ladders by electrophoresis in gels of compounds that have unusual gelling properties, including gelation when the temperature is raised. In addition to its use for the sequencing aspect of the human genome initiative, the data generated will contribute to understanding fundamental characteristics of gel-forming polymers.

New procedures are needed to increase the readable length of a DNA sequencing ladder. Current procedures are limited primarily by resolution of the longer DNA fragments. The PI has developed a procedure for progressive increase in the resolution between DNA bands. No known limit to the achievable resolution exists. Resolution is progressively increased by back and forth motion of DNA; no increase in the length of the apparatus is required. Back and forth motion is induced by a pulsed electrical field; the pulses are orders of magnitude longer than previously used pulses. The resolution of DNA bands increases during both forward and reverse motion (bidirectional increase in resolution). Bidirectional increase in resolution is the cornerstone of the proposed work. Bidirectional increase in resolution for DNA sequencing is best achieved in a capillary format. The following are the specific aims: (a) Tests will be made of the effectiveness of bidirectional increase in resolution for increasing the readable length of a DNA sequencing ladder during capillary-based DNA sequencing. (b) Procedures for bidirectional increase in resolution will be optimized. The criterion for optimization will be reading of the longest possible DNA sequencing ladder in the shortest possible time. Preliminary experiments suggest that exploitation of anti- band broadening effects of pulsed fields will make a significant contribution. Achievement of the specific aims will improve both de novo sequencing and mutation.detection sequencing for all genomes.

Description: (Verbatim from the applicant's abstract) Metabolism-driven change in the conformation of double-stranded DNA is studied to understand several biological events. These events include the packaging of a double-stranded DNA genome in the preformed protein container (capsid) of a bacteriophage. The DNA packaging substrate for some bacteriophages is an end-to-end multimer (concatemer) of the mature genome. The concatemer is cleaved to genomes during packaging. Understanding of these metabolic changes has not been achieved conventionally. One reason is that any given macroscopically-observed change in DNA conformation is unsynchronized and, possibly, variable among microscopically-observed single DNA molecules. Another reason is that these changes occur in complex systems that may consist of more than one phase. The limitations of conventional procedures can be bypassed by using single molecule-fluorescence microscopy. Metabolic events are imaged directly at a resolution of approximately 250 nm. Higher resolution information is obtained by more indirect use of the data from fluorescence microscopy. The PI's laboratory has recently solved the problems that had previously prevented use of this strategy. Both an initiating cleavage of concatemers and entry of DNA into capsids have been observed by singe-molecule fluorescence microscopy. The following are the specific aims of the current proposal: (1) The behavior of single capsids will be observed by fluorescence microscopy before the initiation of packaging of concatemer-associated genomes of bacteriophage T7. Determination will be made of whether capsids search for initiation sites by use of an ATP-driven motor. (2) The formation of the initiation complex will be observed by single-molecule fluorescence microscopy. A hypothesis that proposes initiation via capsid-induced cyclization will be tested. (3) The entry of DNA into a capsid will be observed while the capsid is attached to a solid support. Hypotheses for the mechanism of pumping DNA into a capsid will undergo preliminary tests. (4) Procedures will be further developed for the purpose of analyzing DNA metabolism by single-molecule fluorescence microscopy. The research proposed constitutes a new strategy for understanding the biochemistry of complex systems. This research also reveals the biological and physical constraints that were present when these systems evolved. The procedures and concepts developed will assist in the analysis of all biological systems. They also have use in both environmental biology and disease diagnosis.

DESCRIPTION (provided by applicant): Major current limitations of large-scale genome sequencing can be overcome by achieving the following objectives: (1) increasing the readable length of a DNA sequencing ladder, and (2) incorporating error-flagging-correcting into the process of analyzing a DNA sequencing ladder. The proposed research will achieve both these objectives by use of capillary DNA sequencing. The first objective will be achieved by increasing the effective length of the path of electrophoresis, without increasing the physical length of the capillary. A DNA sequencing ladder will be analyzed by use of several stages (analysis-stages) of conventional capillary electrophoresis. An enhancement-stage will be inserted between each neighboring pair of analysis-stages. During an enhancement-stage, a pulsed electrical field is used to improve the resolution of the unanalyzed portion of the DNA sequencing ladder, while the DNA molecules migrate slightly in a direction opposite to the direction of an analysis-stage. This experimentally-proven strategy (called cyclic capillary electrophoresis) length-resolves DNA fragments that are compressed during an uninterrupted constant field electrophoresis. DNA peaks do not broaden during cyclic capillary electrophoresis. The cyclic character opens the possibility of error-flagging by analyzing the same DNA sequencing fragments more than once. The current data suggest that dramatic advances remain to be made. Needed equipment-software has recently been developed by the PI and his collaborators. Thus, the specific aims are the following: (1) we will develop optimized procedures for using cyclic capillary electrophoresis to increase the readable length of a DNA sequencing ladder. We will test the effects of embedding additional pulses within the pulses of enhancement-stages. (2) We will investigate the use of cyclic electrophoresis for error flagging-correcting during capillary DNA sequencing. (3) We will develop a user-friendly interface for integrating cyclic electrophoresis with current procedures of DNA sequencing. The interface will provide access to different adaptations of cyclic capillary electrophoresis. Each adaptation will help solve a current genomic problem, for example, either closing of a partially assembled genome sequence or performing high accuracy analysis of mutations.

This project partners the University of Texas Health Science Center at San Antonio (UTHSCSA) and the University of Texas at San Antonio (UTSA), in the south Texas region, for connectivity to an advanced network. Southwest Research Institute (SwRI) will serve as the sponsor for UTHSCSA and UTSA and will provide experienced technical and engineering staff to assist in connecting to the Abilene network through the Austin POP via a DS-3 line. UTHSCSA and UTSA have a combined annual research budget of over 113 million dollars, enroll over 21,000 students and both are Hispanic-serving institutions with 56% female enrollment.

The proposed high-speed network will enhance collaborations with scientists at 21 other Abilene-connected institutions and will immediately support eleven research projects via Abilene:
University of Texas Health Science Center at San Antonio
Global Analysis of Hydrodynamic and Thermodynamic Properties of Macromolecules
Development of a Remote NMR Data Acquisition Infrastructure
Single Molecule Video Microscopy
High-Speed Data Sharing of Brainmap Images
University of Texas at San Antonio
GEneral NEural SImulation System (GENESIS) Software System
Analysis of the information content of cortical waves in the turtle visual system
IP-friendly Quality-of-Service Mechanisms
Adaptive Transport Protocols for High-Speed Networks
A Tool for Distance Learning
Learning Community Across Campuses and Institutions
Grid Computation Analysis of Uncertainty Management in the Design and
Construction of Blast Resistance Structures for Anti-Terrorist Applications

These research projects range from strictly biological (e.g., biochemistry at the level of single molecules) to strictly computer science (e.g. network transport protocols that adapt rapidly to the available network capacity). Between the extremes are interdisciplinary research projects at the interface of biological science and mathematics (e.g., general neural simulation system, probabilistic reference system for the human brain, and numerical modeling of a turtle's visual system) plus education of future teachers (e.g., development of teaching strategies and the application of technology).

Abstract Not Provided.

[unreadable] DESCRIPTION (provided by applicant): ATP-driven biological motors are essential to the complex biochemistry of both eukaryotes and prokaryotes. The Pl's research focuses on understanding bacteriophage DNA packaging motors because these motors provide advantages during biochemical/biophysical analysis. A bacteriophage DNA packaging motor is a multimolecular ring attached to an icosahedral protein shell (capsid). A central hole in the ring is a channel through which a double-stranded DNA molecule is motor-driven into the capsid during DNA packaging. Central questions for all biological motors are the following: Is the motor's cycling rate feedback regulated? What is the source of the directional bias of the motor, given the small size and, therefore, high thermal motion of components? What are the details of the motor's cycle? The PI has proposed a detailed hypothesis that has potential answers to these questions. This hypothesis states that a DNA packaging motor has a cycling rate that is feedback regulated. Furthermore, this hypothesis states that the motor is an osmotic pressure gradient-assisted device that has the capacity to be a thermal ratchet. The Specific Aims are the following: (1) The Pl's hypothesis will be tested directly in several aspects. Tests will be performed for whether the motor's cycle is feedback regulated. Tests will also be performed for osmotic pressure-derived DNA packaging force. (2) The various states of a cycle of the DNA packaging motor will be identified and characterized. Recently discovered states of variable capsid size and permeability will be investigated. Analysis will be performed of the temporal relationship among intermediate states during a cycle of a DNA packaging motor. Some analyses will be performed by single-particle fluorescence microscopy. The DNA packaging motors to be studied are those of bacteriophages f29, T3 and T7. The data to be obtained will answer central questions about biological motors, whether or not the Pl's hypothesis is correct. Analysis of bacteriophage DNA packaging motors is expected to provide a basis for understanding the role of motors in disease, especially disease caused by a virus that has a DNA packaging motor. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Single-particle fluorescence microscopy of biological assembly reactions is emerging in its potential to obtain information that is lost during the ensemble averaging of conventional biochemical analysis. However, efforts to realize this potential have stalled at the level of the specimens used. The PI's laboratory has been developing the needed specimen preparation and accompanying procedures. For continuous real time single-particle visualization of assembly, the PI's laboratory has found specimen preparation procedures for concentrating/restricting comparatively large (-30 nm in radius) particles in a thin zone of solution next to a cover glass. However, similar conditions for smaller particles (single protein molecules) are not yet developed. The specific aims are the following: (1) For single-particle fluorescence microscopy, specimen preparation procedures will be developed that achieve (a) concentration of comparatively small particles, including monomeric proteins, in a thin zone of solution next to a cover glass, and (b) retention by these particles of thermal motion. (2) Procedures of single-particle fluorescence microscopy will be developed for observing and analyzing both dimerization and more complex events of the assembly of macromolecules. In so doing, determination will be made of whether our single-particle procedure yields dissociation constants that agree with the dissociation constants measured by use of conventional, ensemble averaging procedures. Analysis will be performed of complex viral assembly. Achievement of these aims will make the assembly of multimolecular complexes routinely accessible to analysis by single-particle fluorescence microscopy. Applications are anticipated in the analysis of the assembly of disease-causing viruses and determining pathways of oncogenesis. [unreadable] [unreadable] [unreadable]

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