Faculty Bibliography

Rapid and reliable detection of mutations at the genetic level is an integral part of modern molecular diagnostics. These mutations can range from dominant single nucleotide polymorphisms within specific loci to codominant heterozygotic insertions and they present considerable challenges to investigators in developing rapid nucleic acid-based amplification assays that can distinguish wild-type from mutant alleles. The recent improvements of real-time polymerase chain reaction (PCR) using self-reporting fluorescence probes have given researchers a powerful tool in developing assays for mutation detection that can be multiplexed for high-throughput screening of multiple mutations and cost effectiveness. Here we describe an application of a multiplexed real-time PCR assay using Molecular Beacon probes for the detection of mutations in codon 54 of the CYP51A gene in Aspergillus fumigatus conferring triazole resistance.

Over the past decade the adoption and refinement of the GAL4 system by the Drosophila field has resulted in a wide array of tools with which the researcher can drive transgene expression in a precise spatiotemporal pattern. The GAL4 system relies on two components: (1) GAL4, a transcriptional activator from yeast, which is expressed in a tissue-specific manner and (2) a transgene under the control of the upstream activation sequence that is bound by GAL4 (UASG). The two components are brought together in a simple genetic cross. In the progeny of the cross, the transgene is only transcribed in those cells or tissues expressing the GAL4 protein. Recent modifications of the GAL4 system have improved the control of both the initiation and the spatial restriction of transgene expression. Here we describe the GAL4 system highlighting the properties that make it a powerful tool for the analysis of gene function in Drosophila and higher organisms.

Abundant epidemiological data are now available (2008) on the human lung cancer response for lifetime radon gas exposure to residential concentrations of 100 Bq m(-3), equal to 22 working level months over 40 y. We combined published pooled epidemiological data and dosimetric calculations of alpha particle hits to target basal or mucous cell nuclei in bronchial epithelium. This yields an estimate that about 10,000 basal nuclei (target) cell hits per cm2 per person over a lifetime are involved in radon-related lung cancer. The DNA target cell area (cross section) for a hit is about 2 bp. The present epidemiology indicates that 1000 persons need to be exposed to this hit rate for observable cancers to be detected. The mechanism proposed is that the extensive prior DNA damage in smokers, followed by alpha particle damage to a critical site in checkpoint genes, accounts for the greater lung cancer response in smokers

Successful completion of fertilization in mammals is dependent on three membrane fusion events. These are (1) the acrosome reaction of sperm, (2) the fusion of sperm and egg plasma membranes to form a zygote, and (3) the cortical reaction of fertilized eggs. Extensive research into the molecular basis of each of these events has identified candidate proteins and factors involved in fusion of membranes during the mammalian fertilization process. Some of this information is provided here.

The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters

Germ cells are the ultimate stem cells because they have the potential to give rise to a new organism. Specified during early embryogenesis in most species, germ cells evade somatic differentiation by using mechanisms such as transcriptional silencing and translational control (Seydoux and Braun 2006; Cinalli et al. 2008). To identify germ-line targets of translational regulation and to understand their mechanism of regulation, we used publicly available databases to identify RNAs localized to germ plasm. Using a transgenic reporter assay, we find that these germ-line RNAs are both spatially and temporally regulated during both oogenesis and embryogenesis by their 3'-untranslated regions (3'UTRs) (Rangan et al. 2008). We find that many RNAs that are spatially and temporally regulated in the early embryo are also translationally regulated during oogenesis. However, RNAs that are similarly regulated during oogenesis are no longer coregulated during embryogenesis, demonstrating that cis-acting sequences within a single RNA are used differentially during the life cycle of the germ line. Our study emphasizes a multifaceted role of translational regulation in germ cells. Many aspects of cellular behavior are shared between germ cells and other stem cells; thus, analysis of the translational regulatory networks controlling translation during the germ-line life cycle may reveal important general features of RNA regulation in stem cells

INTRODUCTION: Bcl-2 antanogene-1 (Bag-1) binds the anti-apoptotic mediator Bcl-2, and enhances its activity. Bcl-2 and Bag-1 are associated with chemotherapy resistance in cancer cells. Drugs that target Bcl-2 are currently in clinical development. The purpose of the present study was to examine expression patterns of Bag-1 in a large cohort of breast tumors and to assess the association with Bcl-2, estrogen receptor, progesterone receptor and Her2/neu, and other clinical/pathological variables. METHODS: Tissue microarrays containing primary specimens from 638 patients with 10-year follow-up were employed, and the expression of Bag-1, Bcl-2, estrogen receptor, progesterone receptor and Her2/neu was assessed using our automated quantitative analysis method. We used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and we measured biomarker expression within the mask using Cy5 conjugated antibodies. RESULTS: High Bcl-2 expression was associated with improved survival in the entire cohort and in the node-positive subset (P = 0.008 and P = 0.002, respectively). High Bag-1 expression was associated with improved survival in the node-positive subset (P = 0.006). On multivariable analysis, neither Bcl-2 nor Bag-1 retained their independence as prognostic markers. Strong associations were found between Bag-1, Bcl-2, estrogen receptor and progesterone receptor. CONCLUSION: Bag-1 and Bcl-2 expression in breast tumors is associated with improved outcome and steroid receptor positivity. Evaluation of Bcl-2 and Bag-1 expression in breast cancer may identify a subset of patients with a favorable prognosis, who might not benefit from chemotherapy or who might benefit from Bcl-2 targeting agents in addition to antihormonal therapy

We report the solid-phase synthesis of novel 2'P-RNA probes for use in fluorescence polarization (FP) ligand binding assays that screens for inhibitors of the yeast 2'- phosphotransferase Tpt1p. The probe was synthesized by utilizing silyl phosphoramidite chemistry and a phosphoramidite synthon containing an orthogonal (DMT) protecting group at its 2'-position. Regioselective removal of the 2'-DMT group and phosphitylation of the unmasked 2'-hydroxyl group afforded the desired 2'P-RNA sequence