Characterization of cis-acting replication control elements in ribosomal RNA genes of Tetrahymena thermophila

This dissertation describes the identification and characterization of specific cis-acting replication control elements in the replication origin of Tetrahymena rDNA. Earlier studies of a mutant rDNA allele, C3-rmm1, identified a cis-acting sequence (the Type I repeat) within the 5[superscript]\u27 non-transcribed spacer of rDNA that affects rDNA replication (Larson et al. 1986). Characterization of two additional, independently-isolated mutants (C3-rmm3 and C3-rmm4) and a revertant of C3-rmm4 strongly argue that the Type I repeat is indeed a cis-acting replication control element. Mutations causing rDNA replication defects were identified in copies of the Type I repeat that are separated by \u3e600 bp. Therefore, the rDNA replication origin may be much larger than typical origins defined in prokaryotes. In addition, the mutation in C3-rmm3 is located within the rRNA gene transcriptional promoter region, suggesting a possible dual role for the Type I repeat in transcription and replication. Together these results provide genetic evidence for a novel sequence-specific element that affects eukaryotic DNA replication;Chromosomal origins of replication in higher eukaryotes appear to share common modular sequence elements (Benbow et al. 1992). Several of these modular sequence elements were found clustered in multiple copies within the origin region of Tetrahymena rDNA. Furthermore, an intrinsically bent DNA structure and nuclear matrix-associated regions were directly demonstrated in the origin region. Replicative intermediates of rDNA that contain bubble structures within the 5[superscript]\u27NTS were detected;A high resolution method for mapping sites of replication initiation was developed. Individual DNA molecules were tagged with streptavidin-gold and visualized using atomic force microscopy. This work provides the basis for a novel technique to examine the first nucleotides incorporated into replicating rDNA

BRST-antifield-treatment of metric-affine gravity

The metric-affine gauge theory of gravity provides a broad framework in which gauge theories of gravity can be formulated. In this article we fit metric-affine gravity into the covariant BRST--antifield formalism in order to obtain gauge fixed quantum actions. As an example the gauge fixing of a general two-dimensional model of metric-affine gravity is worked out explicitly. The result is shown to contain the gauge fixed action of the bosonic string in conformal gauge as a special case.Comment: 19 pages LATEX, to appear in Phys. Rev.

Visualization of circular DNA molecules labeled with colloidal gold spheres using atomic force microscopy

We have imaged gold‐labeled DNA molecules with the atomic force microscope(AFM). Circular plasmid DNA was labeled at internal positions by nick‐translation using biotinylated dUTP. For visualization, the biotinylated DNA was linked to streptavidin‐coated colloidal gold spheres (nominally 5 nm diam) prior to AFM imaging. Reproducible images of the labeled DNA were obtained both in dry air and under propanol. Height measurements of the DNA and colloidal gold made under both conditions are presented. The stability of the DNA‐streptavidin colloidal gold complexes observed even under propanol suggests that this labeling procedure could be exploited to map regions of interest in chromosomal DNA

Imaging Biological Samples with the Atomic-Force Microscope

The application of atomic force microscopy (AFM) to biological investigation is attractive for a number of reasons. Foremost among these is the ability of the AFM to image samples, even living cells, under near native conditions and at resolution equal to, or exceeding, that possible by the best light microscopes. Moreover, the ability of the AFM to manipulate samples it images provides a novel and far reaching application of this technology

Characterization of cis-acting replication control elements in ribosomal RNA genes of Tetrahymena thermophila

This dissertation describes the identification and characterization of specific cis-acting replication control elements in the replication origin of Tetrahymena rDNA. Earlier studies of a mutant rDNA allele, C3-rmm1, identified a cis-acting sequence (the Type I repeat) within the 5[superscript]' non-transcribed spacer of rDNA that affects rDNA replication (Larson et al. 1986). Characterization of two additional, independently-isolated mutants (C3-rmm3 and C3-rmm4) and a revertant of C3-rmm4 strongly argue that the Type I repeat is indeed a cis-acting replication control element. Mutations causing rDNA replication defects were identified in copies of the Type I repeat that are separated by >600 bp. Therefore, the rDNA replication origin may be much larger than typical origins defined in prokaryotes. In addition, the mutation in C3-rmm3 is located within the rRNA gene transcriptional promoter region, suggesting a possible dual role for the Type I repeat in transcription and replication. Together these results provide genetic evidence for a novel sequence-specific element that affects eukaryotic DNA replication;Chromosomal origins of replication in higher eukaryotes appear to share common modular sequence elements (Benbow et al. 1992). Several of these modular sequence elements were found clustered in multiple copies within the origin region of Tetrahymena rDNA. Furthermore, an intrinsically bent DNA structure and nuclear matrix-associated regions were directly demonstrated in the origin region. Replicative intermediates of rDNA that contain bubble structures within the 5[superscript]'NTS were detected;A high resolution method for mapping sites of replication initiation was developed. Individual DNA molecules were tagged with streptavidin-gold and visualized using atomic force microscopy. This work provides the basis for a novel technique to examine the first nucleotides incorporated into replicating rDNA.</p

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA.

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661-670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c-myc proto-oncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found

Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA

We have postulated that chromosomal replication origin regions in eukaryotes have in common clusters of certain modular sequence elements (Benbow, Zhao, and Larson, BioEssays 14, 661–670, 1992). In this study, computer analyses of DNA sequences from six origin regions showed that each contained one or more potential initiation regions consisting of a putative DUE (DNA unwinding element) aligned with clusters of SAR (scaffold associated region), and ARS (autonomously replicating sequence) consensus sequences, and pyrimidine tracts. The replication origins analyzed were from the following loci: Tetrahymena thermophila macronuclear rDNA gene, Chinese hamster ovary dihydrofolate reductase amplicon, human c- myc protooncogene, chicken histone H5 gene, Drosophila melanogaster chorion gene cluster on the third chromosome, and Chinese hamster ovary rhodopsin gene. The locations of putative initiation regions identified by the computer analyses were compared with published data obtained using diverse methods to map initiation sites. For at least four loci, the potential initiation regions identified by sequence analysis aligned with previously mapped initiation events. A consensus DNA sequence, WAWTTDDWWWDHWGWHMAWTT, was found within the potential initiation regions in every case. An additional 35 kb of combined flanking sequences from the six loci were also analyzed, but no additional copies of this consensus sequence were found.This article is from Nucleic Acids Research 22 (1994): 2479, doi: 10.1093/nar/22.13.2479. Posted with permission.</p

Identification of DNA-binding proteins that recognize a Conserved Type I repeat sequence in the replication origin region of Tetrahymena rDNA

An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila. Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo. Fe(ll)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells. Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays. One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences. In contrast, a single-stranded DNA-binding protein, ssATIBF, specifically recognized the A-rich strand of the Type I repeat sequence. Deletion of the 5′ or 3′ borders of the conserved sequence significantly reduced binding of ssA-TIBF. The binding properties of ssATIBF, coupled with genetic evidence that Type I sequences function as c/s-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena.This article is from Nucleic Acids Research 22 (1994): 4432, doi: 10.1093/nar/22.21.4432. Posted with permission.</p