Jeffrey H Miller - US grants

The basic objective of this program is to understand in detail mutational processes, including the mechanism by which mutagens induce mutations and also the pathways that result in spontaneous mutations. Towards this end we have developed and are continuing to develop systems in E. coli which allow the determination of mutagenic specificity at a high degree of resolution. We have extended this work to permit the analysis of mutations induced in mammalian cells by characterizing lacI mutations generated on SV40-based shuttle vectors. By inducing mutations in human cell lines, and then recovering the DNA and transforming E. coli, one can take advantage of the high resolution bacterial genetic systems for the detection and characterization of mutations. We intend to examine mutations induced by different mutagens, and to utilize additional vectors that allow the employment of different human cell lines, such as those from patients with XP, or with Bloom's Syndrome. Because of the presence of carcinogens in the environment, it is of practical importance to understand the manner in which carcinogenic agents cause mutations that can lead to cancer. We are investigating the specificity of mutations induced by UV in human cells, and characterizing the action of oxidative-type mutagens in bacteria, among other carcinogens. The research plan combines genetic and biochemistry, in that mutations are analyzed with a variety of genetic systems prior to selecting representative mutations to analyze by DNA sequencing. In addition to analyzing lacI nonsense mutations, we are employing selection systems in lacI that are specific for each type of base substitution, and also for +1 and -1 frameshifts. By understanding the specificity of mutagenesis, we hope to be able to pinpoint specific premutational lesions and to elucidate the pathways that lead to mutations. Analysis of repair deficient strains, mutator strains and antimutator strains should help identify pathways involved in generating spontaneous mutations.

This proposal is aimed at constructing synthetic tRNA nonsense suppressor genes, which will direct the in vivo synthesis of a series of nonsense (in most cases UAG) condon. The goal is to derive a set of suppressors capable of inserting each of the 20 amino acids at a UAG site. This would greatly aid systematic studies of amino acid substitutions in proteins, since UAG mutations can be introduced by in vivo and in vitro methods at any position in a protein. The information gained from virtual unlimited amino acid substitutions would facilitate protein engineering experiments. The synthetic genes are put together from a series of oligonucleotides and introduced into E. coli cells by a plasmid vector. Additional modifications are introduced by site-directed mutagenesis, by random mutagenesis followed by selection, and by constructing hybrids of different tRNA molecules. The amino acid inserted by each suppressor is determined by protein sequencing in a system that employs an amber mutation in the gene encoding dihydrofolate reductase. Mutants will be sought which improve the specificity of certain suppressors by limiting the mischarging by non-cognate synthetases, such as glutamine tRNA synthetase. Different vector systems will be used to carry the suppressor genes to increase the general utility of the suppressors to different investigators. The goal of this proposal is to facilitate the engineering of specific amino acid replacements in proteins. In principle, new amino acids can be inserted at any site in the product of a cloned gene by a chemical procedure known as site-directed mutagenesis. However, this procedure becomes laborious when large numbers of specific replacements are needed. In collaboration with Dr. John Abelson at the California Institute of Technology, Dr. Miller intends to circumvent this problem by constructing transfer RNA genes that will perform the substitutions in vivo. The transfer RNA genes, known as suppressors, will be constructed so as to allow insertion of any one of the 20 different amino acids whenever the nonsense codon UAG occurs. Since this codon can be created at any location in a cloned gene, virtually all amino acid replacements will be possible. This collection of suppressor genes will be extremely useful to scientists studying the relationships between the structure and function of proteins. Important information on the functioning of transfer RNA molecules will also be obtained.

DESCRIPTION (Adapted from the investigators abstract): The goal of this project is to increase the understanding of protein structure function relationships, with the long term objective of eventually being able to design drugs that specifically inhibit certain enzymes, as well as redesigning enzymes to perform specific functions. The ability to synthesize better inhibitors would have significant impact on therapeutic approaches to different diseases. The specific aim here is to study the structure-function relationships in the E. coli lac repressor and in both E. coli and human thymidylate synthetase, which has been cloned and expressed in E. coli. The approach to be used takes advantage of recent technological advances that make possible the creation of a very large number of amino acid replacements in a protein by employing synthetic nonsense suppressors operating at nonsense sites. It is now possible to insert 13 different amino acids, in series, at each position in a protein corresponding to a UAG (amber) codon, by using a bank of E. coli nonsense suppressors, many of which have been produced by gene synthesis in vitro. Each amber suppressor inserts one specific amino acid at an amber site. By constructing a series of amber mutations in the gene of choice, using both in vivo and in vitro mutagenesis techniques, a virtual systematic replacement of amino acids can be effected. The effects of the replacements on protein function will be measured and considered in terms of the structural alteration in the protein. These comparisons will be aided by the three-dimensional (3D) structures of the respective proteins. The 3D structure of E. coli thymidylate synthetase in combination with substrate and cofactor inhibitors has been solved, and both the human thymidylate synthetase and the E. coli lac repressor have been crystallized in a form that will lead to the solution of their 3D structures. Additional experiments, including the use of second-site revertants, will be employed to pinpoint important contacts in the protein, as will attempts to change the specificity of ligand binding for each protein under study.

DESCRIPTION: The long-term objective of this project is to understand the underlying mechanisms of mutagenesis and define the mutational pathways and repair systems operating in living cells. Because of the relationship between mutagenesis and carcinogenesis, it is the contention of Dr. Miller that this project has a direct bearing on human health. The specific aims of the application involve detecting and characterizing different mutator genes in bacteria, and finding and characterizing their homologs in human cDNA and human genomic libraries. Initially, the work will focus on four mutators which were discovered during the previous funding period. The mutY and mutM genes encode proteins that counteract the effects of the oxidation product 8-oxodG. Work is already ongoing with a fragment of the mutY homolog from a human cDNA library, and this fragment will be used to determine the sequence of the entire gene, detect the genomic segment it is carried on, and to map the gene on the human chromosome. Ultimately, it is planned to screen human tumor lines that are mutators for defects in this gene, and others that can be detected which are homologous to bacterial mutators. Additional studies will focus on the mutA and mutC genes which Dr. Miller has found to create mutations by a novel pathway. A missense suppressor is created that inserts glycine at the aspartic acid codon and, in doing so, probably creates a mutator polymerase by substituting in a fraction of the population, a glycine for an essential aspartic acid in the epsilon subunit of polymerase III. It is contended that this pathway of mutagenesis in bacteria has possible implications for understanding contributions to aging and possibly cancer susceptibility in humans.

DESCRIPTION (adapted from investigator's abstract): It is proposed to construct a series of hybrid microorganisms by combining segments of the genomes of two or more organisms into one single microorganism chromosome. Previous work in this area has involved bringing one or several genes into higher cells, such as the construction of a transgenic mouse, or else bringing one or several genes into a microorganism via a plasmid vector that remains in the cytoplasm. It is proposed here, however, to create new microorganisms by introducing large (greater than 10 genes, and as many as 100 to 1,000 genes) genetic segments into the cell and incorporating them directly into the chromosome by genetic engineering. In many cases the constructed hybrids will have a series of desired properties derived from different microorganisms, such as nitrogen fixation pathways, oil slick degradation pathways, and antibiotic synthesis pathways. These new species of microorganisms can be constructed in either a planned, directed manner, or by random combination of segments of different genomes to open up an era of "combinatorial genomes."

DESCRIPTION (provided by applicant): Numerous members of the third kingdom, Archaea, are extreme hyperthermophiles. Such organisms pose many interesting fundamental questions in molecular biology, since all of their life processes occur at elevated temperatures. For instance, how do regulatory proteins recognize and bind to operators at temperatures above 100 degrees C? How can the DNA replicate with high fidelity, and how do these organisms avoid excessive mutation due to heat-induced DNA lesions, and what repair strategies do they employ? We propose to develop a functional genomic analysis of one of the high temperature archaea, Pyrobaculum aerophilum, that can grow up to 104 degrees C, with an optimal growth temperature of 100 degrees C. We have sequenced and fully annotated the 2.2 mega base genome of Pyrobaculum aerophilum, and are completing the construction of microarrays containing every ORF, in collaboration with Todd Lowe at Stanford/U.C. Santa Cruz. We will carry out experiments on genome-wide gene expression in different environments, and use the data to identify coregulated genes. Through extended analyses, we hope to use the data to set up experiments to identify regulatory pathways and regulatory proteins. We will extend the development of a genetic system with the aim of being able to carry out reverse genetics in this organism. We have begun an indepth investigation of the repair systems in this organism, and we will continue to characterize DNA repair strategies and proteins.

This proposal is for a conference grant for the sixth Small Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 20,24, 1998, at the conference center in Lake Arrowhead, California. There is great excitement over the prospect of comparing the sequences and contents of as many as 25 microbial genomes over the next year or two. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. While there have been a number of recent genome conferences, continuing completion and release of new genome sequences and developing continued need of such conference. This meeting has become established as a major annual microbial genomics conference during the last seven years and has assured status and quality.

This proposal is for a conference grant for the eighth Small Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 24-28, 2000, at the conference center in Lake Arrowhead, California. There is great excitement over the prospect of comparing the sequences and contents of as many as 50 microbial genomes over the next year or two. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To acheive these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will also be on genomes of pathogens. While there have been a number of recent genome conferences, continuing completion and release of new genome sequences and developing momentum for analysis of this huge amount of information means the community has continued need of such conferences. This meeting has become established as a major annual microbial genomics conference during the last seven years and has assured status and quality.

This proposal is for a conference grant for the eighth Small Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 24-28, 2000, at the conference center in Lake Arrowhead, California. There is great excitement over the prospect of comparing the sequences and contents of as many as 50 microbial genomes over the next year or two. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will also be on genomes of pathogens. While there have been a number of recent genome conferences, continuing completion and release of new genome sequences and developing momentum for analysis of this huge amount of information means the community has continued need of such conferences. This meeting has become established as a major annual microbial genomics conference during the last seven years and has assured status and quality.

The long term goal of this research program is to uncover and understand mutational pathways, the DNA repair processes that counteract these pathways, and the consequences of repair defects. This proposal is an extension of previous work that defined new "mutator strains" in bacteria that have higher mutation rates than wild-type and that led to the elucidation of the GO system for oxidative damage. The human mutt gene, an essential component of this system, has been cloned. This work has now been extended to include targeted gene knockout mice. This proposal seeks to engineer and characterize a set of knockout mice lacking oxidative repair, as well as mice lacking different combinations of repair enzymes. Lacking certain repair systems can lead to cancer susceptibilities. It is therefore important to determine whether the absence of repair systems for oxidative damage leads to increased cancer rates or other abnormalities, such as accelerated aging. Specifically, the proposed research seeks to do the following. 1. Generate a double knockout mouse of mMYH with a DNA repair gene mOGG1. The OGG1 gene encodes a protein with a function similar to the bacterial MutM protein. Double knockouts might be expected to lack the ability to repair certain types of oxidative damage to the DNA. 2. Characterize mMYH knockout mice and mMYH-/- mOGG1-/- double knockout mice, analyze their phenotype and screen for abnormalities and cancer susceptibilities that might result from the reduced ability to repair oxidative damage. 3. Characterize primary mouse embryonic fibroblasts derived from both MYH-/- and MYH-/- OGG1-/- mice. 4. Generate and characterize additional combinations of mMYH knockout mice with other repair gene or tumor suppressor gene knockouts, such as MSH2 and p53.

DESCRIPTION (provided by applicant): This proposal is for a conference grant for the Tenth Small Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 8-12, 2002, at the UCLA conference center at Lake Arrowhead, California. There is great excitement over the prospect of comparing the sequences and contents of as many as 200 microbial genomes over the 2-3 years. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will also be on genomes of pathogens and biothreats, and a second focus on genome organization, This meeting has become established as a major annual microbial genomics conference during the last nine years and has assured status and quality.

[unreadable] DESCRIPTION (provided by applicant): This proposal is for a conference grant for the Twelfth Microbial Genomes Conference (formerly the E. coli and Small Genomes Conference) to be held September 26-30, 2004, at the UCLA conference center at Lake Arrowhead, California. The genomics landscape has changed now that numerous genomes are available and that microarrays and bioinformatics methods are advancing rapidly. It is a critical time to foster multi-disciplinary approaches to making sense out of all the genomic sequence information. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will also be on microbial communities and biodiversity. An additional focus will be on modeling and constructing circuits.and pathways. This meeting has become established as a major annual microbial genomics conference during the last twelve years and has assured status and quality. [unreadable] [unreadable] [unreadable]

This proposal is for a conference grant for the Fourteenth Microbial Genomics Conference[unreadable] (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) to be[unreadable] held September 24-28, 2006, at the UCLA conference center at Lake Arrowhead, California.[unreadable] The genomics landscape has changed now that numerous genomes are available and that[unreadable] microarrays and bioinformatics methods are advancing rapidly. It is a critical time to foster multidisciplinary[unreadable] approaches to making sense out of all the genomic sequence information. Considerable[unreadable] integration of different aspects of microbiology will undoubtedly take place, including new[unreadable] perspectives on evolution and the requirements for life. To achieve these goals, particularly of[unreadable] integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will[unreadable] include active genome researchers, such as sequencing experts and informatics specialists working[unreadable] in database design, sequence analysis, or simulation; geneticists developing new methods to take[unreadable] advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists[unreadable] who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or[unreadable] gene regulation. One focus of this conference will also be synthetic biology, namely, the modeling[unreadable] and constructing circuits and pathways and generating organisms with new capacities. An additional[unreadable] focus will be on microbial communities and biodiversity, including pathogens and biothreat agents.[unreadable] This has a direct bearing on human health. This meeting has become established as a major annual[unreadable] microbial genomics conference during the last fourteen years and has assured status and quality.

[unreadable] DESCRIPTION (provided by applicant): This proposal is for a conference grant for the Sixteenth Microbial Genomics Conference (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) to be held September 14-18, 2008, at the UCLA conference center at Lake Arrowhead, California. The genomics landscape has changed now that numerous genomes are available and that microarrays and bioinformatics methods are advancing rapidly, and that whole microbial communities are open to investigation at the sequence level. It is timely to foster multi-disciplinary approaches to define and solve problems. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will be on microbial communities and biodiversity, including pathogens and biothreat agents. This has a direct bearing on human health. This meeting has become established as a major annual microbial genomics conference during the last sixteen years and has assured status and quality. This conference deals with the molecular genetics of microorganisms, including pathogens and biothreats. Methods facilitating the detection and fingerprinting of these microorganisms, as well as understanding how their genomes relate to their functions, all topics at the conference, are directly related to human health. [unreadable] [unreadable] [unreadable]

Intellectual Merit: The Eighteenth Microbial Genomics Conference will be held September 12-16, 2010, at the UCLA Conference Center at Lake Arrowhead, California. The meeting is aimed at fostering interactions among scientists from multiple disciplines who are studying various aspects of microbial biology enabled by the wealth of genomic information and bioinformatics technologies developed during recent years. This conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation; geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis; and cell biologists and biochemists who have specialized in basic science areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will be on microbial communities and biodiversity, including the human microbiome (the collection of microorganisms in the human body). Another focus will be on finding new methods of annotation, as the conference includes a workshop on genome annotation.

Broader Impacts: This meeting has become established as a major annual microbial genomics conference during the last eighteen years. It continues to offer a major opportunity for young investigators and students to attend, present their research as posters or talks, and discuss recent findings with leading researchers in the field. This exchange of ideas is critically important to network- and career-building.

DESCRIPTION (provided by applicant): Abstract PI: MILLER, JEFFREY H Project: 1R13AI091176-01 Title: Eighteenth Microbial Genomes Conference Accession Number: 3255477 ================== NOTICE: THIS ABSTRACT WAS EXTRACTED FROM APPLICATION AND HAS NOT BEEN PROOFED BY AN SRA.WHEN THERE ARE PROBLEMS WITH THE APPLICATION SCANNING PROCESS, THE EXTRACTED TEXT MAY BE INCORRECT OR INCOMPLETE. ================== This proposal is for a conference grant for the Eighteenth Microbial Genomics Conference (formerly the E. coli and Small Genomes Conference, and the Microbial Genomes Conference) to be held September 12-15, 2010, at the UCLA Conference Center at Lake Arrowhead, California. The genomics landscape has changed now that numerous genomes are available and that microarrays and bioinformatics methods are advancing rapidly, and that whole microbial communities are open to investigation at the sequence level. It is timely to foster multi-disciplinary approaches to define and solve problems. Considerable integration of different aspects of microbiology will undoubtedly take place, including new perspectives on evolution and the requirements for life. To achieve these goals, particularly of integration and synergy, scientists from diverse disciplines must interact. Thus, this conference will include active genome researchers, such as sequencing experts and informatics specialists working in database design, sequence analysis, or simulation;geneticists developing new methods to take advantage of genomic information, e.g. for functional analysis;and cell biologists and biochemists who have specialized in areas now ripe for genomics such as metabolism, chromosome structure, or gene regulation. One focus of this conference will be on microbial communities and biodiversity, including the human microbiome. A focus will also be on pathogens, including those involved in gastric cancers, and biothreat agents. This has a direct bearing on human health. This meeting has become established as a major annual microbial genomics conference during the last eighteen years and has assured status and quality. This conference deals with the molecular genetics of microorganisms, including pathogens, those that lead to gastric cancer, and biothreats. Methods facilitating the detection and fingerprinting of these microorganisms, as well as understanding how their genomes relate to their functions, and defining the human microbiome, all topics at the conference, are directly related to human health.