Molecular Biology Commons^™

Role Of Cdx4 And Sp5l In Zebrafish Development, Wesley Tsai

Honors Theses

The Caudal Type Homeobox transcription factors cdx are a family of genes found in vertebrates that regulates body regionalization and anterior-posterior patterning. They are also responsible for regulating axial elongation, but the mechanisms behind this behavior are not known. Previous studies in mouse embryonic stem cells have shown that the cdx genes are necessary for upregulating the gene sp5 which may be linked to axial elongation. Sp5 is a zinc-finger transcription factor belonging to the specificity protein (sp) family. Our group has used in-situ hybridization experiments on zebrafish embryos to show that sp5-like (sp5l) is transcribed within tailbud tissues that …

Dpc29 Promotes Post-Initiation Mitochondrial Translation In Saccharomyces Cerevisiae, Kyle A. Hubble, Michael F. Henry

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Mitochondrial ribosomes synthesize essential components of the oxidative phosphorylation (OXPHOS) system in a tightly regulated process. In the yeast Saccharomyces cerevisiae, mitochondrial mRNAs require specific translational activators, which orchestrate protein synthesis by recognition of their target gene's 5'-untranslated region (UTR). Most of these yeast genes lack orthologues in mammals, and only one such gene-specific translational activator has been proposed in humans-TACO1. The mechanism by which TACO1 acts is unclear because mammalian mitochondrial mRNAs do not have significant 5'-UTRs, and therefore must promote translation by alternative mechanisms. In this study, we examined the role of the TACO1 orthologue in yeast. We …

Diversity Of The Major Histocompatibility Complex In African Penguins (Spheniscus Demersus) In Situ, Athena Schalk

Undergraduate Honors Thesis Projects

The Major Histocompatibility Complex (MHC) is responsible for the immune response in all jawed vertebrates and protects individuals against a variety of pathogens and diseases. Maintaining genetic diversity within the MHC exons is critical to protecting endangered species. African penguins (Spheniscus demersus) are in danger of losing their MHC diversity in isolated populations due to reductions in population size associated with environmental changes and human activity. This study analyzes the diversity within the exons in the DNA encoding the MHC by amplifying the exons through polymerase chain reaction and identifying alleles through denaturing gradient gel electrophoresis. Wild populations of …

N-Terminal Domain Of Human Uracil Dna Glycosylase (Hung2) Promotes Targeting To Uracil Sites Adjacent To Ssdna-Dsdna Junctions, Brian P Weiser, Gaddiel Rodriguez, Philip A Cole, James T Stivers

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

The N-terminal domain (NTD) of nuclear human uracil DNA glycosylase (hUNG2) assists in targeting hUNG2 to replication forks through specific interactions with replication protein A (RPA). Here, we explored hUNG2 activity in the presence and absence of RPA using substrates with ssDNA-dsDNA junctions that mimic structural features of the replication fork and transcriptional R-loops. We find that when RPA is tightly bound to the ssDNA overhang of junction DNA substrates, base excision by hUNG2 is strongly biased toward uracils located 21 bp or less from the ssDNA-dsDNA junction. In the absence of RPA, hUNG2 still showed an 8-fold excision bias …

Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried

Center for Structural Biology Faculty Publications

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O⁶-alkylguanine and O⁴-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ω_ave. We found …

Molecular Analysis Confirming The Introduction Of Nile Crocodiles, Crocodylus Niloticus Laurenti 1768 (Crocodylidae), In Southern Florida, With An Assessment Of Potential For Establishment, Spread, And Impacts., Michael R. Rochford, Kenneth L. Krysko, Frank J. Mazzotti, Matthew W. Shirley, Mark W. Parry, Joseph A. Wasilewski, Jeffrey S. Beauchamp, Christpher R. Gillette, Edward F. Metzger Iii, Michiko A. Squires, Louis A. Somma

Papers in Herpetology

The state of Florida, USA, has more introduced herpetofauna than any other governmental region on Earth. Four species of nonnative crocodilians have been introduced to Florida (all since 1960), one of which is established. Between 2000–2014 we field-collected three nonnative crocodilians in Miami-Dade County, Florida, and one in Hendry County, Florida. We used DNA barcoding and molecular phylogenetics to determine species identification and native range origin. Also, we described diet, movement, and growth for one crocodile. Our molecular analyses illustrated that two of the crocodiles we collected are most closely related to Nile Crocodiles (Crocodylus niloticus) from South Africa, suggesting …

Dna Sequencing Activity, Sarah O'Leary-Driscoll

Sequencing & Genome Mining

This activity is meant to introduce students to basic DNA sequencing techniques. Using a hands-on approach helps them understand the fundamentals of what is happening in the lab, and the questions that accompany the activity can lead to more in depth discussions about sequencing, and the development of the next generation sequencing techniques.

Large-Scale Identification Of Chemically Induced Mutations In Drosophila Melanogaster., Nele A Haelterman, Lichun Jiang, Yumei Li, Vafa Bayat, Hector Sandoval, Berrak Ugur, Kai Li Tan, Ke Zhang, Danqing Bei, Bo Xiong, Wu-Lin Charng, Theodore Busby, Adeel Jawaid, Gabriela David, Manish Jaiswal, Koen J T Venken, Shinya Yamamoto, Rui Chen, Hugo J Bellen

Faculty Publications

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of …

Meiotic Dna Re-Replication And The Recombination Checkpoint, Nicole Ann Najor

Wayne State University Dissertations

Progression through meiosis occurs through a strict sequence of events, so that one round of DNA replication precedes programmed recombination and two nuclear divisions. Cyclin dependent kinase 1 (Cdk1) is required for meiosis, and any disruption in its activity leads to meiotic defects. The Cdk1 inhibitor, Sic1, regulates the G1-S transition in the mitotic cell cycle and the analogous transition in meiosis. We have employed a form of Sic1, Sic1deltaP^HA, that is mutated at multiple phosphorylation sites and resistant to degradation. Meiosis specific expression of Sic1deltaP^HA disrupts Cdk1 activity and leads to significant accumulation of over replicated …

Electroporation-Mediated Delivery Of A Naked Dna Plasmid Expressing Vegf To The Porcine Heart Enhances Protein Expression, W. G. Marshall Jr., B. A. Boone, J. D. Burgos, S. I. Gografe, M. K. Baldwin, M. L. Danielson, M. J. Larson, D. R. Caretto, Y. Cruz, B. Ferraro, L. C. Heller, K. E. Ugen, M. J. Jaroszeski, R. Heller

Bioelectrics Publications

Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly …

Mechanism Of High-Mobility Group Protein B Enhancement Of Progesterone Receptor Sequence-Specific Dna Binding, James S. Adelman, Sarah C. Roemer, Mair E.A. Churchill, Dean P. Edwards

James S. Adelman

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE …

Cationic Surfactant Mediated Hybridization And Hydrophobization Of Dna Molecules At The Liquid/Liquid Interface And Their Phase Transfer, Murali Sastry, Ashavani Kumar, Mrunalini Pattarkine, Vidya Ramakrishnan, Krishna N. Ganesh

Faculty Works

Hybridization of complementary oligonucleotides mediated by a cationic surfactant at the water/hexane interface leads to hydrophobic, double-helical DNA which may be readily phase transferred to the organic phase and cast into thin films on solid substrates.

Molecular Cloning And Rare Cleavage Mapping Of Human 2p, 6q, 8q, 12q, And 18q Telomeres, Roberto A. Macina, Ken Morii, Xue-Lan Hu, Dimitri G. Negorev, Chrysanthe Spais, Lisa A. Ruthig, Harold C. Riethman

Medical Diagnostics & Translational Sciences Faculty Publications

Large terminal fragments of human chromosomes 2p, 6q, 8q, 12q, and 18q were cloned using yeast artificial chromosomes (YACs). RecA-assisted restriction endonuclease (RARE) cleavage analysis of genomic DNA samples from 11 unrelated individuals using YAC-derived probes confirmed the telomeric localizations of the half-YACs studied. The cloned Fragments provide telomeric closure of maps for the respective chromosome arms and will supply the reagents needed for analyzing and sequencing these distal subtelomeric regions.

An Analysis Of Mitochondrial Dna In Rett Syndrome And Other Neurodegenerative Disorders, Catherine Erickson Burgess

Theses and Dissertations in Biomedical Sciences

Mitochondrial dysfunction resulting from mutations on mitochondrial DNA (mtDNA) is being recognized in a growing spectrum of diseases. These diseases, resulting from single base mutations, large deletions, or insertions, have been largely neuromuscular in origin. However, as an understanding of the effects of mtDNA mutations progresses, attention is now focusing on neurodegenerative diseases. Rett Syndrome (RS), a progressive neurodegenerative disease with predominantly female cases, demonstrates morphologic mitochondrial changes, mitochondrial enzyme deficiencies and maternal inheritance (characteristic of mtDNA diseases). No investigation of mtDNA involvement has been previously conducted and, to date, no biological marker exists for this disorder.

Our preliminary studies …

The Characterization Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Vicky L. Montoya, Wendy L. Murdoch, Roy C. Ogle, John L. Keating, Robert M. Grainger

Medical Diagnostics & Translational Sciences Faculty Publications

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they …

The Purification Of Ribosomal Rna Gene Chromatin From Physarum Polycephalum, Sally A. Amero, Roy C. Ogle, John L. Keating, Vicky L. Montoya, Wendy L. Murdoch, Robert M. Grainger

Medical Diagnostics & Translational Sciences Faculty Publications

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then …