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PREFERENTIAL INHIBITION OF THE GROWTH OF VIRUS-TRANSFORMED CELLS IN CULTURE BY RIFAZONE-82, A NEW RIFAMYCIN DERIVATIVE

Description: Rifazone-8{sub 2} (R-8{sub 2}), a new rifamycin derivative, is shown to preferentially inhibit the growth of virus-transformed chick cells in culture. Macromolecular synthesis and glucose uptake of transformed cells are also appreciably decreased in the presence of low concentrations of R-8{sub 2} where the normal cells appear unaffected. While R-8{sub 2} is shown to be a selective inhibitor of RNA-directed DNA polymerase in vitro, its action on the growth of transformed cells may involve some other mechanism.
Date: March 28, 1974
Creator: Bissell, Mina J.; Hatie, Carroll; Tischler, Allan N. & Calvin, Melvin.
Partner: UNT Libraries Government Documents Department

CYTOCIDAL EFFECT OF RIFAMYCIN DERIVATIVES ON ASCITES TUMOR CELLS: STUDIES WITH 125I-IODODEOXYURIDINE

Description: We have previously reported the chemotherapeutic effect of rifamycin derivatives on an ascites tumor, using increased life span as a criterion. These derivatives inhibit (1) RNA-instructed DNA polymerase in crude viral extracts; (2) virus-induced transformation in tissue cultures; and (3) the growth of tumors in vivo. One rifamycin derivative, rifazone-8{sub 2} (R-8{sub 2}), not only inhibits transformation in chick fibroblasts but affects the growth of transformed cells. The present study demonstrates that rifampicin and R-8{sub 2} act as cytocidal (rather than cytostatic) agents against some ascites cell lines.
Date: August 1, 1978
Creator: Hughes, Ann M. & Calvin, Melvin
Partner: UNT Libraries Government Documents Department

THE EFFECT OF RIFAMPICIN, AND TWO DERIVATIVES, ON CELLS INFECTEDWITH MOLONEY SARCOMA VIRUS

Description: It is shown that rifampicin, and especially its relative dimethyl-N-benzyl-N-desmethyl rifampicin, can inhibit focus formation by Moloney sarcoma virus on BALB/3T3 tissue cultures. At a dose level of 10 {micro}g/ml DMB appears to totally inhibit focus formation while reducing virus replication by at least a factor of fifty and cell proliferation by only a factor of three. These observations, taken together with those of others, suggest a role for the hybrid RNA-DNA dependent DNA polymerase and the gene for its synthesis both in normal cell processes and in the transformation process.
Date: March 1, 1971
Creator: Calvin, Melvin.; Joss, Urs R.; Hackett, Adeline J. & Owens, RobertB.
Partner: UNT Libraries Government Documents Department

A NON-CLEAVABLE UmuD VARIANT THAT ACTS AS A UmuD' MIMIC

Description: UmuD{sub 2} cleaves and removes its N-terminal 24 amino acids to form UmuD'{sub 2}, which activates UmuC for its role in UV-induced mutagenesis in E. coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD has been shown to bind to the beta processivity clamp (''beta'') of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif (14-TFPLF-18) in UmuD and showed that it has a moderate influence on UV-induced mutagenesis but is required for the cold sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K{sub d} values as determined by changes in tryptophan fluorescence. However, this data also implies that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is noncleavable, we find that cells harboring it exhibit phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic.
Date: October 26, 2005
Creator: Beuning, P J; Simon, S M; Zemla, A; Barsky, D & Walker, G C
Partner: UNT Libraries Government Documents Department

Single molecule study of a processivity clamp sliding on DNA

Description: Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.
Date: July 5, 2007
Creator: Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A et al.
Partner: UNT Libraries Government Documents Department

Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

Description: Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.
Date: January 1, 1995
Creator: Mishra, N.C.
Partner: UNT Libraries Government Documents Department

PROTEOLYTIC REMOVAL OF THE CARBOXYL TERMINUS OF THE T4 GENE 32 HELIX-DESTABILIZING PROTEIN ALTERS THE T4 IN VITRO REPLICATION COMPLEX

Description: The proteolytic removal of about 60 amino acids from the COOH terminus of the bacteriophage T4 helix-destabilizing protein (gene 32 protein) produces 32*I, a 27,000-dalton fragment which still binds tightly and cooperatively to single-stranded DNA. The substitution of 32*I protein for intact 32 protein in the seven-protein T4 replication complex results in dramatic changes in some of the reactions catalyzed by this in vitro DNA replication system, while leaving others largely unperturbed. (1) Like intact 32 protein, the 32*I protein promotes DNA synthesis by the DNA polymerase when the T4 polymerase accessory proteins (gene 44/62 and 45 proteins) are also present. The host helix-destabilizing protein (Escherichia coli ssb protein) cannot replace the 32*I protein for this synthesis. (2) Unlike intact 32 protein, 32*I protein strongly inhibits DNA synthesis catalyzed by the T4 DNA polymerase alone on a primed single-stranded DNA template. (3) Unlike intact 32 protein, the 32*I protein strongly inhibits RNA primer synthesis catalyzed by the T4 gene 41 and 61 proteins and also reduces the efficiency of RNA primer utilization. As a result, de novo DNA chain starts are blocked completely in the complete T4 replication system, and no lagging strand DNA synthesis occurs. (4) The 32*I protein does not bind to either the T4 DNA polymerase or to the T4 gene 61 protein in the absence of DNA; these associations (detected with intact 32 protein) would therefore appear to be essential for the normal control of 32 protein activity, and to account at least in part for observations 2 and 3, above. We propose that the COOH-terminal domain of intact 32 protein functions to guide its interactions with the T4 DNA polymerase and the T4 gene 61 RNA-priming protein. When this domain is removed, as in 32*I protein, the helix destabilization induced by the protein is controlled ...
Date: July 1, 1980
Creator: Burke, R.L.; Alberts, B.M. & Hosoda, J.
Partner: UNT Libraries Government Documents Department

Regulation of chloroplast number and DNA synthesis in higher plants. Final report, August 1995--August 1996

Description: The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focused on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The research focused on the isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.
Date: June 17, 1997
Creator: Mullet, J.E.
Partner: UNT Libraries Government Documents Department

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing. Final report, June 1, 1988--January 31, 1996

Description: This project has focused on the DNA polymerase of phage T7 for use in DNA sequencing. A complex of T7 DNA polymerase and E. coli thioredoxin form a highly processive DNA polymerase. The exonuclease activity of the enzyme can be reduced by chemical or genetic modifications resulting in an enzyme that has several properties useful in sequencing including high processivity and lack of discrimination against dideoxynucleotides. Manganese ion eliminates all discrimination against ddNTPs allowing sequence determination based on band intensity. A single tyrosine residue in the active site of T7 DNA polymerase is responsible for the efficient incorporation of ddNMPs. Replacement of the phenylalanine at this position in Klenow or Taq DNA polymerase with tyrosine eliminates discrimination against ddNTPs, a property that has advantages for cycle sequencing. Pyrophosphorolysis catalyzed by a polymerase results in the hydrolysis of specific fragments in DNA sequencing reactions, a problem that is eliminated by the addition of pyrophosphatase. The thioredoxin domain of gene 5 protein has been identified and transferred to Klenow DNA polymerase to make it processive. We have crystallized a complex of T7 DNA polymerase/thioredoxin bound to a primer-template in the presence of a dNTP.
Date: August 1, 1996
Creator: Richardson, C.C.
Partner: UNT Libraries Government Documents Department

PCR detection of groundwater bacteria associated with colloidal transport

Description: Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research.
Date: February 29, 1996
Creator: Cruz-Perez, P.; Stetzenbach, L. D. & Alvarez, A. J.
Partner: UNT Libraries Government Documents Department

Characterization of the mammalian DNA polymerase gene and protein. Annual progress report

Description: Methods were developed to purify the DNA polymerases of the {alpha}-family from Chinese hamster cells and their mutants selected as resistant to aphidicolin or specific inhibitor of DNA polymerases of the {alpha}-family. The wild type and mutant DNA polymerases were characterized with respect to their biochemical properties. A methodology was also developed to identify the replication intermediates and aphidicolin was found to inhibit a replication intermediate of the 24Kb size indicating the fact that aphidicolin inhibits the elongation process during DNA replication. This is the first demonstration of such role of aphidicolin in the eukaryotic DNA replication.
Date: January 1, 1993
Creator: Mishra, N.C.
Partner: UNT Libraries Government Documents Department

Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

Description: Consistent with the long term goal of our research to understand the nature of the key enzymes in eukaryotic DNA replication we have characterized the properties of the wild type DNA polymerases of the {alpha}-family and their mutants. We have also provided evidence for the role of aphidicolin in the elongation process of the in vivo DNA replication in eukaryotic cells. We also developed a technology for planned prep from a large numbers of clones for direct screening by size or restriction digestion in order to facilitate our goals to clone the DNA polymerase gene.
Date: January 1, 1994
Creator: Mishra, N.C.
Partner: UNT Libraries Government Documents Department

Influence of Translation Initiation on Organellar Protein Targeting in Arabidopsis

Description: A primary focus of the Mackenzie laboratory is the elucidation of processes and machinery for mitochondrial genome maintenance and transmission in higher plants. We have found that numerous organellar DNA maintenance components in plants appear to be dual targeted to mitochondria and plastids. Of particular interest was the observation that some twin (tandemly arrayed) dual targeting presequences appeared to utilize non-AUG alternative translation initiation, allowing for multiple translation starts at a single gene. Two aspects of this phenomenon were of particular interest: (1) Alternative translation initiation might provide a mechanism to regulate protein targeting temporally and spatially, a possibility that had not been demonstrated previously, and (2) alternative translation initiation might occur in genes involved in nuclear-controlled mitochondrial genome recombination, thought to be exclusively mitochondrial in their function. During the course of this research, we pursued three aims, with an emphasis on two specific genes of interest: POLgamma2, an organellar DNA polymerase, and MSH1, a MutS homolog thought to participate in mitochondrial, but not plastid, genome recombination surveillance. Our aims were to (1) Identify additional genes within Arabidopsis and other genomes that employ non-AUG alternative translation initiation, (2) Locate sequences upstream to the annotated AUG that confer alternative non-AUG translation initiation activity, and (3) Identify cis and trans factors that influence start site selection in genes with non-AUG starts. Toward these ends, we have shown that non-AUG initiation occurs in a number of genes, likely influencing targeting behavior of the protein. We have also shown that start site selection is strongly influenced by Kozak consensus sequence environment, indicating that alternative translation initiation in plants occurs by relaxation of ribosome scanning.
Date: April 18, 2011
Creator: Mackenzie, Sally A.
Partner: UNT Libraries Government Documents Department

Structure/Function Analysis of DNA-glycosylases That Repair Oxidized Purines and Pyrimidines and the Influence of Surrounding DNA Sequence on Their Interactions

Description: The overall goal of this project was to elucidate the structure/function relationships between oxidized DNA bases and the DNA repair enzymes that recognize and remove them. The NMR solution structure of formamidopyrimidine DNA glycosylase (Fpg) that recognizes oxidized DNA purines was to be determined. Furthermore, the solution structures of DNA molecules containing specific lesions recognized by Fpg was to be determined in sequence contexts that either facilitate or hinder this recognition. These objectives were in keeping with the long-term goals of the Principal Investigator's laboratory, that is, to understand the basic mechanisms that underpin base excision repair processing of oxidative DNA lesions and to elucidate the interactions of unrepaired lesions with DNA polymerases. The results of these two DNA transactions can ultimately determine the fate of the cell. These objectives were also in keeping with the goals of our collaborator, Dr. Michael Kennedy, who is studying the repair and recognition of damaged DNA. Overall the goals of this project were congruent with those of the Department of Energy's Health Effects and Life Sciences Research Program, especially to the Structural Biology, the Human Genome and the Health Effects Programs. The mission of the latter Program includes understanding the biological effects and consequences of DNA damages produced by toxic agents in the many DOE waste sites so that cleanup can be accomplished in a safe, effective and timely manner.
Date: August 22, 2005
Creator: Wallace, Susan S.
Partner: UNT Libraries Government Documents Department

Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments

Description: Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using ?29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2 percent genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9 percent of the sequences had significant similarities to known proteins, and ''clusters of orthologous groups'' (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible.
Date: December 10, 2005
Creator: Abulencia, C.B.; Wyborski, D.L.; Garcia, J.; Podar, M.; Chen, W.; Chang, S.H. et al.
Partner: UNT Libraries Government Documents Department

Measurement of mutation and repair in mammalian cells/action of specific mutagens and antimutagens/genome exposure reaction in cancer and other disease conditions. Final subcontract report, April 1, 1996- March 31, 1996

Description: This is the final report for the project dealing with the the measurement of mutation and repair in mammalian cells, action of specific mutagens and antimutagens, and genome exposure reaction in cancer and other disease conditions. The overall objectives of this research are threefold: to develop and improve methodology for measurement of mutation and repair in mammalian cells and to apply it to measurement of the effectiveness of mutagens, antimutagens, and other molecules to as to achieve greater power in prevention of cancer and genetic disease; to analyze theoretically and experimentally the action of specific mutagens and antimutagens; and to investigate the role of genome exposure reaction in cancer and other disease conditions to secure improve preventive and treatment modalities.
Date: September 1, 1996
Creator: Puck, T.T.
Partner: UNT Libraries Government Documents Department

Base sequence effects on DNA replication influenced by bulky adducts. Final report, March 1, 1995--February 28, 1997

Description: Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that are present in air, food, and water. While PAH compounds are chemically inert and are sparingly soluble in aqueous solutions, in living cells they are metabolized to a variety of oxygenated derivatives, including the high mutagenic and tumorigenic diol epoxide derivatives. The diol epoxides of the sterically hindered fjord region compound benzo[c]phenanthrene (B[c]PhDE) are among the most powerful tumorigenic compounds in animal model test systems. In this project, site-specifically modified oligonucleotides containing single B[c]PhDE-N{sup 6}-dA lesions derived from the reactions of the 1S,2R,3R,4S and 1R,2S,3S,4R diol epoxides of B[c]PhDE with dA residues were synthesized. The replication of DNA catalyzed by a prokaryotic DNA polymerase (the exonuclease-free Klenow fragment E. Coli Po1 I) in the vicinity of the lesion at base-specific sites on B[c]PhDE-modified template strands was investigated in detail. The Michaelis-Menten parameters for the insertion of single deoxynucleotide triphosphates into growing DNA (primer) strands using the modified dA* and the bases just before and after the dA* residue as templates, depend markedly on the stereochemistry of the B[c]PhDE-modified dA residues. These observations provide novel insights into the mechanisms by which bulky PAH-DNA adducts affect normal DNA replication.
Date: May 31, 1997
Creator: Geacintov, N. E.
Partner: UNT Libraries Government Documents Department

[Studies of the repair of radiation-induced genetic damage in Drosophila]. Annual progress report, June 1, 1992--June 30, 1993

Description: The most interesting discovery made over the past year derives from sequence analysis of cDNAs from the putative mus308 gene. The theoretical translation product of this gene contains a DNA polymerase domain near the carboxy terminus and DNA/RNA helicase motifs near the amino terminus. There is currently no precedent in the literature for a single polypeptide containing both of these domains. The protein appears to be a novel DNA repair enzyme which should be fruitful ground for future enzymological analysis. The authors have identified two ORFs by sequence analysis of the transforming fragment containing the mei-41 gene and of corresponding cDNAs. ORF 1 includes the P element insertion sites and encodes a peptide of 757 amino acids. ORF 2 starts 900 base pairs downstream of ORF 1 and encodes a peptide of 1,037 amino acids. This putative peptide shows homology to the yeast DNA repair genes, rad50 of S. cerevisiae and rad3 of S. pombe.
Date: September 1, 1998
Partner: UNT Libraries Government Documents Department

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

Description: This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity of these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.
Date: December 31, 1993
Creator: Richardson, C.C.
Partner: UNT Libraries Government Documents Department

Regulation of chloroplast number and DNA synthesis in higher plants. Final report

Description: The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.
Date: November 10, 1995
Creator: Mullet, J.E.
Partner: UNT Libraries Government Documents Department

Final report [DNA Repair and Mutagenesis - 1999]

Description: The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.
Date: May 30, 2001
Creator: Walker, Graham C.
Partner: UNT Libraries Government Documents Department

[Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

Description: This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.
Date: December 31, 1992
Partner: UNT Libraries Government Documents Department

Viruses of eukaryotic green algae. Final technical report, June 1, 1989--February 1, 1992

Description: We have isolated and partially characterized many large, polyhedral, DNA containing, plaque forming viruses which infect certain unicellular, eukaryotic, chlorella-like green algae. These viruses have several unique features, including the fact that they code for DNA site-specific endonucleases and DNA methyltransferases. The primary objectives of this study were to identify, clone, and characterize some of the virus-encoded DNA methyltransferases and DNA restriction endonucleases in order to understand their biological function.
Date: December 31, 1992
Creator: Van Etten, J. L.
Partner: UNT Libraries Government Documents Department