Ken Hampel | University of Vermont (original) (raw)

Papers by Ken Hampel

Nucleic Acids Research, 1989

Biophysical Journal, Feb 1, 2011

Abstract Increasingly, the experience and knowledge gleaned via research-based interrogation of t... more Abstract Increasingly, the experience and knowledge gleaned via research-based interrogation of the human genome is being leveraged and translated for use in the clinical realm to inform medical decision making. The clinical implementation of genome-based diagnostics is fraught with a variety of challenges that include but are not limited to the scope of: knowledge and the medical significance of genome-based data, scalable technology and infrastructure, informatics, and regulatory requirements. This chapter will provide an overview of state-of-the-art genomic technologies with a focus on DNA-based Next-Generation Sequencing. Herein is a review of the clinical applications of genomic testing and the common considerations for developing this testing in the clinical laboratory setting. The common challenges to incorporating genomics into clinical care for the laboratory, clinicians, and patients are also addressed.

Molecular Immunology, Oct 1, 1995

A large number of phosphorothioate DNAs and mixed ribo/deoxyribo duplexes were prepared and their... more A large number of phosphorothioate DNAs and mixed ribo/deoxyribo duplexes were prepared and their immunogenicity was studied in mice. Only those polymers which were nucleaseresistant were immunogenic and in these cases monoclonal antibodies were prepared. The specificity of the antibodies was measured by direct and competitive Solid Phase Radioimmune Assay (SPRIA) and on this basis four types of antibody could be identified. Type I antibodies are specific for the immunizing polymer and show very limited crossreactivity. For example, Jel 384 binds only to poly(dsA).poly(dT); Jel 453 and 462 bind only to poly(dsG).poly(dC)and poly(dsG).poly(dm'C). Type II antibodies bind to most polymers containing the appropriate modification but will not bind to unmodified DNAs. For example, Je1343 binds to most thio DNAs regardless of sequence; Jel346 binds well to most ribose-containing polymers and may be a useful reagent for the detection of the 'A' family of conformations. Type III antibodies bind to most nucleic acids whether modified or not. Their specificities are similar to autoimmune antibodies. Type IV antibodies are single strand-specific such as Jel 383 which binds to poly(dT). There were no examples of antibodies which bound specifically to the immunizing DNA and the unmodified polymer. Thus, modified DNAs cannot be used to prepare sequence-specific reagents. Also, the immunogenicity of modified nucleic acids may limit their usefulness in antisense technologies.

Biochemistry, May 29, 2009

The glmS ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located ups... more The glmS ribozyme is a conserved riboswitch in numerous Gram-positive bacteria and is located upstream of the glucosamine-6-phosphate (GlcN6P) synthetase reading frame. Binding of GlcN6P activates site-specific self-cleavage of the glmS mRNA, resulting in the downregulation of glmS gene expression. Unlike other riboswitches, the glmS ribozyme does not undergo structural rearrangement upon metabolite binding, indicating that the metabolite binding pocket is preformed in the absence of ligand. This observation led us to test if individual steps in the reaction pathway could be dissected by initiating the cleavage reaction before or after Mg 2+-dependent folding. Here we show that self-cleavage reactions initiated with simultaneous addition of Mg 2+ and GlcN6P are slow (3 min-1) compared to reactions initiated by addition of GlcN6P to glmS RNA that has been prefolded in Mg 2+-containing buffer (72 min-1). These data indicate that some level of Mg 2+-dependent folding is rate-limiting for catalysis. Reactions initiated by addition of GlcN6P to the prefolded ribozyme also resulted in a 30-fold increase in the apparent ligand K d compared to those of reactions initiated by a global folding step. Time-resolved hydroxyl-radical footprinting was employed to determine if global tertiary structure formation is the rate-limiting step. The results of these experiments provided evidence for fast and largely concerted folding of the global tertiary structure (>13 min-1). This indicates that the rate-limiting step that we have identified either is a slow folding step between the fast initial folding and ligand binding events or represents the rate of escape from a nativelike folding trap. † K.M.B. was supported by Cancer Biology Training Grant CA09286/ 22-26 from the National Cancer Institute.

Journal of Bacteriology, Jun 29, 2012

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the os... more Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used 13 C nuclear magnetic resonance (13 C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.

Biochemistry, Jun 1, 2006

We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of... more We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of methods with the aim of evaluating the magnitude of RNA conformational change induced by binding of the cofactor, glucosamine 6-phosphate (GlcN6P). Hydroxyl radical footprinting of a trans-acting ribozyme complex identifies several sites of solvent protection upon incubation of the RNA in Mg(2+)-containing solutions, providing initial evidence of the tertiary fold of the ribozyme. Under these folding conditions and at GlcN6P concentrations that saturate the ligand-induced cleavage reaction, we do not observe changes to this pattern. Cross-linking with short-wave UV light of the complex yielded similar overall results. In addition, ribozyme-substrate complexes cross-linked in the absence of GlcN6P could be gel purified and then activated in the presence of ligand. One of these active cross-linked species links the base immediately 3&amp;amp;amp;#39; of the cleavage site to a highly conserved region of the ribozyme core and could be catalytically activated by ligand. Combined with recent studies that argue that GlcN6P acts as a coenzyme in the reaction, our data point to a riboswitch mechanism in which ligand binds to a prefolded active site pocket and assists in catalysis via a direct participation in the reaction chemistry, the local influence on the geometry of the active site constituents, or a combination of both mechanisms. This mode of action is different from that observed for other riboswitches characterized to date, which act by inducing secondary and tertiary structure changes.

Experimental and Molecular Pathology, Apr 1, 2017

Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard... more Targeted genomic profiling (TGP) using massively parallel DNA sequencing is becoming the standard methodology in clinical laboratories for detecting somatic variants in solid tumors. The variety of methodologies and sequencing platforms in the marketplace for TGP has resulted in a variety of clinical TGP laboratory developed tests (LDT). The variability of LDTs is a challenge for test-to-test and laboratory-to-laboratory reliability. At the University of Vermont Medical Center (UVMMC), we validated a TGP assay for solid tumors which utilizes DNA hybridization capture and complete exon and selected intron sequencing of 29 clinically actionable genes. The validation samples were run on the Illumina MiSeq platform. Clinical specificity and sensitivity were evaluated by testing samples harboring genomic variants previously identified in CLIA-approved, CAP accredited laboratories with clinically validated molecular assays. The Molecular Laboratory at Dartmouth Hitchcock Medical Center (DHMC) provided 11 FFPE specimens that had been analyzed on AmpliSeq Cancer Hotspot Panel version 2 (CHPv2) and run on the Ion Torrent PGM. A Venn diagram of the gene lists from the two institutions is shown. This provided an excellent opportunity to compare the inter-laboratory reliability using two different target sequencing methods and sequencing platforms. Our data demonstrated an exceptionally high level of concordance with respect to the sensitivity and specificity of the analyses. All clinically-actionable SNV and InDel variant calls in genes covered by both panels (n = 17) were identified by both laboratories. This data supports the proposal that distinct gene panel designs and sequencing workflows are capable of making consistent variant calls in solid tumor FFPE-derived samples.

Biochemistry and Cell Biology, Mar 1, 1993

The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresi... more The mobility of yeast chromosomes was analysed by two-dimensional pulsed-field gel electrophoresis. The first dimension was run at pH 8.0 in a 1% agarose gel. In the second dimension the electrophoresis conditions were identical, except that the pH was lowered and ethidium, spermine, or ionic detergents were added. Any mobility changes between the two dimensions could be identified as a deviation from the diagonal. At pH 6.0 the mobility of the chromosomes increases severalfold, whereas at pH 4.5 none of the chromosomes move into the agarose gel. The pH-induced mobility changes were reversed by the addition of 2 μg/mL of ethidium or 1% lauryl sarcosine. Alternatively, spermine at 1 μM enhanced the pH-mediated mobility changes. Hysteresis was also evident, since upon lowering the pH to 4.5 and then running the gel at pH 7 the mobilities were decreased. These results are interpreted in terms of pH-mediated triplex formation which causes chromosome condensation and thus mobility shifts. The effects of pH are reversed by ethidium which destabilizes triplexes, but enhanced by spermine which favours triplex formation. Therefore, chromosomes may be capable of spontaneous condensation which is mediated by tertiary interactions between appropriate duplex DNA sequences.Key words: triplex DNA, pulsed-field gel electrophoresis, chromosome structure, DNA mobility, chromosome condensation.

Biochemistry, May 17, 1994

Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobas... more Linear plasmids were constructed containing two pyrimidine tracts that were 0.34 and 0.94 kilobases (kb) from either end and were separated by 2.8 kb. The tracts [d(TCCTTC), and d(CTTCCT), where n = 6 or 121 were designed so as to be able to form triplexes with each other but not with themselves. Upon lowering of the pH to 4 in the presence of spermine, these plasmids form intermolecular dimers and intramolecular loops of 2.8 kb, as judged from mobility changes on agarose gels. A tethered loop could also be formed in a linear plasmid containing two identical tracts by adding an homologous single-stranded oligopyrimidine, but not an oligopurine. In plasmids containing different tracts, the formation of both dimers and loops could be blocked by adding either homologous single-stranded oligopyrimidine but not an oligopurine. Together with the requirement of low pH, these results demonstrate that triplex formation is of the pyr-pur.pyr type. The extent of dimer and loop formation was dependent on the length of the pyrimidine tract; dimers could be detected in plasmids containing the 72 base pair (bp) inserts after incubation at pH 6, but in plasmids containing the 36 bp inserts, a pH of 5 was required. Hysteresis was also evident to a remarkable extent. Once formed at pH 4, loops and dimers remained stable indefinitely at pH 8, suggesting that the structures become topologically trapped. However, the structures were resolved into the component linear plasmids by incubation with nuclease P1. This is the first demonstration of a braided or hydrogen-bonded knot between two linear duplexes and may have implications for chromosomal loop formation.

Journal of Molecular Biology, Sep 1, 1995

such tracts, which could form triplexes with each other but not with themselves, were cloned into... more such tracts, which could form triplexes with each other but not with themselves, were cloned into a plasmid at separate sites. Upon lowering the S7N 0WO pH, linear, open circular and relaxed plasmid molecules formed a number 2 Department of Biochemistry of novel structures that were observed on agarose gels and directly by University of Alberta electron microscopy. In open circles a stable join was formed between the two Pyr•Pur tracts giving rise to molecules resembling dumbells, trefoils and tetrafoils, which collectively are termed T-loops. The structure was stable at pH 8 and contains a single-stranded region that was sensitive to P 1 nuclease. Thus, there is no apparent topological impediment to the formation of triplex-mediated loops in circular molecules. These structures may be important for gene regulation and chromosome condensation.

Journal of Bacteriology, Oct 4, 2013

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundan... more Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa's response to the host, particularly changes regulated by the hostderived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa by using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters, 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes and the BetX and CbcXWV quaternary amine transport proteins. We characterized the GbdR consensus binding site and used it to identify that the recently characterized acetylcholine esterase gene, choE (PA4921), is also regulated by GbdR. The regulon member not directly controlled by GbdR is the secreted lipase gene lipA, which was also the only regulon member repressed under GbdR-activating conditions. Determination of the GbdR regulon provides deeper understanding of how GbdR links bacterial metabolism and virulence. Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites.

Biochemistry, Mar 11, 2011

The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds... more The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine-6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5′UTR of the glmS mRNA, resulting in down regulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg 2+-containing buffers effectively isolates the rapid ligand binding and catalytic events (k obs > 60 min −1) from rate-limiting folding (k obs <4 min −1). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure, but are unable to promote ligand binding and catalysis rates >4 min −1. Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand binding pocket. In further support of this hypothesis, our data show that a non-phosphorylated analog of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity but the rapid initiation of catalysis upon the addition of ligand is stricter.

Biochemistry, Feb 1, 1993

Medical Microbiology and Immunology, Sep 25, 2012

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. T... more Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this

The Journal of Molecular Diagnostics, Nov 1, 2018

Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutatio... more Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutation profiling via next-generation sequencing (NGS). Specimens derived via fine-needle aspiration, a common biopsy technique, are subjected to a variety of cytopreparatory methods compared with surgical biopsies that are almost uniformly processed as formalin-fixed, paraffin-embedded tissue. Therefore, the fine-needle aspirationederived specimens most commonly accepted for molecular analysis are cell blocks (CBs), because they are processed most similarly to surgical biopsy tissue. However, CB preparations are fraught with challenges that risk unsuccessful sequencing and repeat biopsies, with the potential to further increase health care costs and delay clinical care. The diversity of cytopreparations and the resource-intensive clinical validation of NGS pose significant challenges to more consistent use of non-CB (NCB) cytology specimens. As part of clinical validation of a targeted NGS assay, DNA subjected to nine cytopreparatory methods was evaluated for sequencing performance and was shown to be uniformly acceptable for clinical NGS. Of the 379 clinical cases analyzed after validation, the majority (56%) were derived from NCB cytology specimens. This specimen class had the lowest DNA insufficiency rate (1.5%) and showed equivalent sequencing performance to surgical and CB formalinfixed, paraffin-embedded tissue. NCB cytology specimens are valuable sources of tumor nucleic acid and are the preferred specimen type for clinical NGS at our institution.

Biochemistry, May 1, 1991

The stability of triplex D N A was investigated in the presence of the polyamines spermine and sp... more The stability of triplex D N A was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly[d(TC)].poly [d(GA)] showed that at low ionic strength and p H 7, 3 p M spermine was sufficient to cause dismutation of all of Registry No. Poly[d(TC)].poly [d(GA)], 29627-66-5; spermine, 71-44-3; spermidine, 124-20-9.

The EMBO Journal, Nov 15, 2001

The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozy... more The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by a conformational change in which N1 and O6 of G8 become closely apposed to the scissile phosphodiester. UV cross-linking, hydroxyl-radical footprinting and native gel electrophoresis indicate that G8 variants inhibit the reaction at a step following domain association, and that the tertiary structure of the inactive complex is not measurably altered. Rate±pH pro®les and¯uorescence spectroscopy show that protonation at the N1 position of G8 is required for catalysis, and that modi-®cation of O6 can inhibit the reaction. Kinetic solvent isotope analysis suggests that two protons are transferred during the rate-limiting step, consistent with rate-limiting cleavage chemistry involving concerted deprotonation of the attacking 2¢-OH and protonation of the 5¢-O leaving group. We propose mechanistic models that are consistent with these data, including some that invoke a novel keto±enol tautomerization.

Biochemistry, Feb 1, 2001

The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactiv... more The hairpin ribozyme is a small endonucleolytic RNA motif with potential for targeted RNA inactivation. It optimally cleaves substrates containing the sequence 5′-GU-3′ immediately 5′ of G. Previously, we have shown that tertiary structure docking of its two domains is an essential step in the reaction pathway of the hairpin ribozyme. Here we show, combining biochemical and fluorescence structure and function probing techniques, that any mutation of the substrate base U leads to a docked RNA fold, yet decreases cleavage activity. The docked mutant complex shares with the wild-type complex a common interdomain distance as measured by time-resolved fluorescence resonance energy transfer (FRET) as well as the same solvent-inaccessible core as detected by hydroxyl-radical protection; hence, the mutant complex appears nativelike. FRET experiments also indicate that mutant docking is kinetically more complex, yet with an equilibrium shifted toward the docked conformation. Using 2-aminopurine as a site-specific fluorescent probe in place of the wild-type U, a local structural rearrangement in the substrate is observed. This substrate straining accompanies global domain docking and involves unstacking of the base and restriction of its conformational dynamics, as detected by time-resolved 2-aminopurine fluorescence spectroscopy. These data appear to invoke a mechanism of functional interference by a single base mutation, in which the ribozyme-substrate complex becomes trapped in a nativelike fold preceding the chemical transition state.