Ralph Hazlewood and Matthew Jorgenson will present their research on Thursday, December 15th, 2011
Background on Ralphs talk:
Identification and Characterization of Genetic Factors Responsible for Cavitary Optic Disk Anomalies
Glaucoma is the second leading cause of irreversible blindness in America and is characterized as an optic neuropathy with visual field loss and progressive degeneration of the optic nerve seen as increased optic disk cupping. Many cases of glaucoma occur in part due to high intraocular pressure (IOP), but glaucoma can occur at any IOP; glaucoma at normal IOP is called normal tension glaucoma (NTG). One way to study NTG is to investigate similar forms of optic nerve disease that also occur in the absence of elevated IOP. Cavitary optic disc anomalies (CODA) are associated with congenital excavation of the optic nerve that in some patients progressively deteriorate resembling the cupping seen in glaucoma. Based on the similarities between NTG and CODA patients, we are searching for the gene that causes autosomal dominant inheritance of CODA in a large family. Prior linkage studies mapped the CODA gene to a 13.5 Mbp segment of chromosome 12q14 (maximum non-parametric linkage score = 21.7). We have examined the linked region for the gene that causes CODA using comparative genome hybridization (CGH) and expression constructs. Study of affected family members with CODA using CGH identified a copy number variation (CNV) within the previously linked locus. A heterozygous triplication of a 6kb segment was found to be co-inherited with CODA in this pedigree and absent in unaffected family members and in normal controls. Subsequent analysis showed a two-fold increase in expression when the 6kb segment is cloned into a reporter vector and transfected into HEK293T cells. We report a CNV within the previously linked region that is co-inherited with CODA in our family. We hypothesize that this CNV leads to dysregulation of the expression of an extracellular protease gene and ultimately to the development of CODA. Future studies include fluorescent in situ hybridization (FISH), Southern blotting to characterize the triplication and generation of transgenic animals.
Background on Matthew’s talk:
Discovery and characterization of new bacterial cell division genes
Cell division is a process essential to all organisms, yet it remains poorly understood. In the bacterium Escherichia coli, cell division is mediated by the formation of the septal ring at the midcell through the coordinated interaction of at least thirty proteins. Two components of this assembly are FtsE and FtsX, which together comprise a putative ATP-binding cassette transporter. While previous work with FtsEX has demonstrated it is important in maintaining the integrity of the septal ring, its exact biochemical function is unknown. To further characterize ftsEX function in cell division, our lab carried out a screen for multicopy suppressors of an ftsEX null mutant. Two of the plasmids returned in this screen carried a gene for an outer membrane lipoprotein named NlpI and various amounts of flanking DNA. Interestingly, an nlpI mutant has a cell division defect at elevated temperature (Ohara et al. 1999), but the precise function of NlpI is not known.
To show that nlpI is indeed responsible for rescue of the ftsEX mutant, I subcloned nlpI into an expression vector and demonstrated that rescue did not require any DNA outside the nlpI gene. Multicopy nlpI was not able to rescue ftsA(Ts), ftsI(Ts),or ftsZ(Ts) but did show weak suppression of an ftsQ(Ts) mutant. I raised polyclonal antibody against NlpI and used it to show that there are ~2,000 molecules of the protein per cell during growth in LB media. Currently I am conducting a structure-function analysis of the protein that focuses on the importance of localization to the outer membrane and a proposed substrate binding pocket. I am also revisiting the reported phenotypes of an nlpI null mutant in hopes that this will provide some more specific insights into the role of the protein in cell division.