Biochemistry, Biophysics, and Structural Biology Commons^™

Chemical Synthesis Of Sensitive Dna, Komal Chillar

Dissertations, Master's Theses and Master's Reports

Over the past decades, researchers have tried various chemical methods to synthesize modified oligodeoxynucleotides (ODNs, i.e. short segments of DNAs). Traditional ODN synthesis methods require strong basic, and nucleophilic conditions for the deprotection and cleavage of the ODN from the solid support. However, the sensitive ODNs containing labile functionalities are vulnerable to such harsh conditions. Sensitive ODNs have a wide range of applications in research and pharmaceuticals. To synthesize sensitive ODNs, researchers devised different strategies but no practical methods have been developed. To overcome these challenges, we developed alkyl Dim alkyl Dmoc technology. This innovative technology uses weakly basic and …

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 …

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 …

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.

Evolution Of Repetitive Proteins: Spider Silks From Nephila Clavipes (Tetragnathidae) And Araneus Bicentenarius (Araneidae), Richard D. Beckwitt, Steven Arcidiacono, Robert Stote

Richard D Beckwitt

Spider silks are highly repetitive proteins, characterized by regions of polyalanine and glycine-rich repeating units. We have obtained two variants of the Spidroin 1 (NCF-1) silk gene sequence from Nephila clavipes. One sequence (1726 bp) was from a cloned cDNA, and the other (1951 bp) was from PCR of genomic DNA. When these sequences are compared with each other and the previously published Spidroin 1 sequence, there are differences due to sequence rearrangements, as well as single base substitutions. These variations are similar to those that have been reported from other highly repetitive genes, and probably represent the results …

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 …

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 …

Variants And Polymorphisms Of Three Repetitive Dna Families In The Human Genome, Robert M. Roudabush

Electronic Theses and Dissertations

A novel 0.6 kb LINE family in human DNA, designated L2Hs, has been described (Musich and Dykes 1986). Studies employing clone N6.4, containing three 0.6 kb segments of this family, indicate that these sequences are interspersed and moderately repetitive. Two additional variant sequences of the L2Hs family, N6.1 and N6.3, have been identified. Restriction mapping of each cloned segment indicates similarities among N6.4, N6.3 and N6.1. When the cloned DNAs were cleaved with restriction enzymes and subjected to cross-hybridization, each cloned insert produced a pattern indicating that the sequences contained in N6.1 and N6.3 are represented in at least one …

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 …