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 BTBD1 and BTBD2, two similar BTB-domain-containing Kelch-like proteins that interact with Topoisomerase I

BACKGROUND: Two-hybrid screening for proteins that interact with the core domain of human topoisomerase I identified two novel proteins, BTBD1 and BTBD2, which share 80% amino acid identities. RESULTS: The interactions were confirmed by co-precipitation assays demonstrating the physical interaction of BTBD1 and BTBD2 with 100 kDa topoisomerase I from HeLa cells. Deletion mapping using two-hybrid and GST-pulldown assays demonstrated that less than the C-terminal half of BTBD1 is sufficient for binding topoisomerase I. The topoisomerase I sequences sufficient to bind BTBD2 were mapped to residues 215 to 329. BTBD2 with an epitope tag localized to cytoplasmic bodies. Using truncated versions that direct BTBD2 and TOP1 to the same cellular compartment, either the nucleus or the cytoplasm, co-localization was demonstrated in co-transfected Hela cells. The supercoil relaxation and DNA cleavage activities of topoisomerase I in vitro were affected little or none by co-incubation with BTBD2. Northern analysis revealed only a single sized mRNA for each BTBD1 and BTBD2 in all human tissues tested. Characterization of BTBD2 mRNA revealed a 255 nucleotide 90% GC-rich region predicted to encode the N-terminus. BTBD1 and BTBD2 are widely if not ubiquitously expressed in human tissues, and have two paralogs as well as putative orthologs in C. elegans and D. melanogaster. CONCLUSIONS: BTBD1 and BTBD2 belong to a small family of uncharacterized proteins that appear to be specific to animals. Epitope-tagged BTBD2 localized to cytoplasmic bodies. The characterization of BTBD1 and BTBD2 and their interaction with TOP1 is underway

SINE indel polymorphism of AGL gene and association with growth and carcass traits in Landrace × Jeju black pig F2 population

Genetic polymorphisms in the glycogen debrancher enzyme (AGL) gene were assessed with regard to their association with growth and carcass traits in the F2 population crossbred Landrace and Jeju (Korea) Black pig. Three genotypes representing the insertion and/or deletion (indel) polymorphisms of short interspersed nuclear element were detected at frequencies of 0.278 (L/L), 0.479 (L/S), and 0.243 (S/S), respectively. The AGL S allele-containing pigs evidenced significantly heavier body weights at birth, the 3rd week, 10th week, and 20th week during developmental stages and higher average daily gains during the late period than were noted in the L/L homozygous pigs (P < 0.05), respectively. However, average daily gains during the early period were not significantly associated with genotype distribution (P > 0.05). With regard to the carcass traits, the S allele pigs (S/-) evidenced significantly heavier carcass weights and thicker backfat than was measured in L/L homozygous pigs (P < 0.05). However, body lengths, meat color, and marbling scores were all found not to be statistically significant (P > 0.05). Consequently, the faster growth rate during the late period and backfat deposition rather than intramuscular fat deposition cause differences in pig productivity according to genotypes of the AGL gene. These findings indicate that the AGL genotypes may prove to be useful genetic markers for the improvement of Jeju Black pig-related crossbreeding systems

The SR Protein B52/SRp55 Is Required for DNA Topoisomerase I Recruitment to Chromatin, mRNA Release and Transcription Shutdown

DNA- and RNA-processing pathways are integrated and interconnected in the eukaryotic nucleus to allow efficient gene expression and to maintain genomic stability. The recruitment of DNA Topoisomerase I (Topo I), an enzyme controlling DNA supercoiling and acting as a specific kinase for the SR-protein family of splicing factors, to highly transcribed loci represents a mechanism by which transcription and processing can be coordinated and genomic instability avoided. Here we show that Drosophila Topo I associates with and phosphorylates the SR protein B52. Surprisingly, expression of a high-affinity binding site for B52 in transgenic flies restricted localization, not only of B52, but also of Topo I to this single transcription site, whereas B52 RNAi knockdown induced mis-localization of Topo I in the nucleolus. Impaired delivery of Topo I to a heat shock gene caused retention of the mRNA at its site of transcription and delayed gene deactivation after heat shock. Our data show that B52 delivers Topo I to RNA polymerase II-active chromatin loci and provide the first evidence that DNA topology and mRNA release can be coordinated to control gene expression

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