2004 Pilot Projects

Title: Engineered Antisense Gene Attenuation in Streptomyces coelicolor
Investigator(s): Brian O. Bachmann, Department of Chemistry
Description: In this in vivo "knock-down" experiment Dr. Bachmann's lab is investigating, for the first time, the ability of antisense RNA to function in trans in actinomycetes. Once developed, this methodology can be used to rapidly interrogate the function of cryptic biosynthetic genes in a variety of actinomycetes and other prokaryotes involved in biosynthesis and environmental toxicology. The model system used, Streptomyces coelicolor A3(2), has been shown to produce two pigmented antibiotics, a blue-pigmented polyketide actinorhodin and a bright red tripyrrole prodigiosin. Several antisense sequence delivery vectors were constructed containing actinomycete specific promoters and transcriptional terminators for the purpose of scanning for antisense sequences. An actinorhodin synthesis pathway-specific transcriptional regulator and a prodigeosin specific biosynthetic gene were amplified from S. coelicolor A3(2) genomic DNA and introduced into the antisense delivery vectors. The expression competence of the vectors was tested by transforming them into a mutant strain in which the two genes were knocked out by double-crossover homologous recombination. Recovery strains were obtained, verifying the competence of antisense delivery vectors in S. coelicolor A3(2).

Title: Analysis of Mechanisms Regulating the Sensing and Repair of Stalled Replication Forks
Investigator(s): David K. Cortez, Department of Biochemistry
Description: Stalling and/or collapse of DNA replication forks in difficult-to-replicate chromosomal regions or at DNA lesions is common, especially in cells with defects in cell cycle checkpoint proteins such as ATR. The ATR kinase is required to sense stalled replication forks and to coordinate DNA repair, fork stabilization/re-initiation, and cell cycle transitions. The goal of Dr. Cortez's project has been to establish a method of stalling a single replication fork at a defined locus in human cells to facilitate experiments aimed at determining how ATR and other checkpoint proteins promote resolution of stalled replication forks and prevent genomic instability. Dr. Cortez's lab has successfully used triplex forming oligonucleotides conjugated to an interstrand crosslinking agent to block replication of an episomal vector in human cells. They have achieved approximately 70% efficiency of inducing an interstrand crosslink in the DNA prior to transfection into human cells. This crosslinked episome is unable to replicate efficiently. Furthermore, an increase has been observed in the monoubiquitinated form of the FANCD2 protein in cells transfected with the crosslinked plasmid. FANCD2 monoubiquitination is an early step in the cellular response to replication stress.

Title: Proteomic Analysis of Axonal Degeneration
Investigator(s): William M. Valentine, Department of Pathology
Description: This project was undertaken to determine the utility of proteomic methods for delineating changes in protein expression and post-translational modifications in Schwann cells and axons undergoing axonal degeneration. Experiments were conducted to assess the sequence of changes in the proteome of peripheral nerve undergoing axonal degeneration in a nerve crush injury model using 2D difference gel electrophoresis (DIGE) as a function of time subsequent to injury. Experiments optimizing methods for isolation of protein from peripheral nerve and their analysis using 2D DIGE were also performed, which demonstrated the presence of a substantial population of proteins in the basic range. Nerve injury surgeries and tissue collections of injured and control nerve were completed, and the samples were prepared for analysis by 2D DIGE.