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Biomedical subjects

William A Bonass

Publications and source records attributed to William A Bonass.

7 recordsLinked to original sources
PubMed2006

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

DNA↗
PubMed2006

Studying silane mobility on hydrated mica using ambient AFM.

The mobility of the mono-functional aminosilane, aminopropyldimethylmethoxysilane (APDMMS), on mica has been investigated under ambient relative humidity (RH) using tapping-mode (TM) AFM. Silane layers were formed from vapour phase at various, controlled humidities and then imaged at ambient laboratory conditions (typically 30-40% RH). At low RH of formation (<25%) films without any resolvable sub-structure were formed, and these were stable to the imaging probe. At high RH of formation (>25%), where islands of phase II water are known to exist on mica, a two-phase domain structure was seen as two height levels and the surface area of the higher domains correlated with the RH. Sequential images taken over a 30-60 min time period show that these domains are mobile and at intermediate to high RH the domains coalesce under the influence of the scanning AFM tip. The results suggest the APDMMS resides at the air-water interface (rather than interacting with the mica) and that it preferentially interacts with the mobile phase II water as opposed to the phase I water tightly bound to the mica.

Journal Article↗
PubMed2006

Imaging RNA polymerase-amelogenin gene complexes with single molecule resolution using atomic force microscopy.

The AMELX gene encoding the enamel matrix protein, amelogenin, is located within (and in the opposite orientation to) the first intron of the ARHGAP6 gene, which encodes a GTPase-activating protein. The orientation of these two genes with respect to each other raises the possibility that they may undergo simultaneous convergent transcription during amelogenesis. The aim of this study was to use atomic force microscopy (AFM) to study a transcriptionally active amelogenin DNA template and to investigate the binding of RNA polymerase to convergently aligned promoters. Images of RNA polymerases stalled on DNA templates were obtained following incubation of the template with RNA polymerases and ribonucleotide triphosphates. A linear DNA template incorporating an intact rat amelogenin cDNA flanked by convergently aligned coliphage T7 and T3 promoters was constructed and shown to be transcriptionally active in vitro. Atomic force microscopy images of transcription complexes revealed globular structures, corresponding to single RNA polymerase molecules bound at specific locations on the DNA templates. These results indicate that AFM allows the visualization of individual RNA polymerases on DNA templates, offering a realistic approach to investigating the concept of convergent transcription of nested genes, which may lead to an understanding of whether the simultaneous expression of AMELX and ARHGAP6 is possible during the formation of tooth enamel.

Amelogenesis↗
PubMed2005

Formation of aminosilane-functionalized mica for atomic force microscopy imaging of DNA.

Factors affecting the functionalization of mica with aminosilanes, in particular, aminopropyltriethoxysilane (APTES-mica), formed from the vapor phase have been systematically studied. The relative humidity (RH) during vapor deposition has been varied, and postdeposition treatment through baking has been used, as well as the comparison of mono and trifunctionality, to investigate how optimal surfaces for AFM imaging of DNA are formed. It is found that the stability of the APTES layers is a consequence of lateral polymerization and not covalent attachment to the mica substrate. At low RH (<25%), DNA adopts an open, well-resolved conformation, whereas at >25% RH, DNA surface-induced condensation occurs. Contact mode AFM scratching experiments show that two main structures of the silane layer exist at different humidity: a monolayer exists at RH < 25%, and a bilayer structure exists at RH > 25%. Finally, structural changes that these two layer types undergo after baking at 150 degrees C were investigated by AFM and X-ray photoelectron spectroscopy (XPS), and these now prevented DNA from binding to the APTES-mica, except in the presence of Mg(II) ions.

Aluminum Silicates↗
PubMed2003

Hydrostatic pressure modulates proteoglycan metabolism in chondrocytes seeded in agarose.

OBJECTIVE: To investigate the effect of isolated hydrostatic pressure on proteoglycan metabolism in chondrocytes. METHODS: Bovine articular chondrocytes cultured in agarose gels were subjected to 5 MPa hydrostatic pressure for 4 hours in either a static or a pulsatile (1 Hz) mode, and changes in glycosaminoglycan (GAG) synthesis, hydrodynamic size, and aggregation properties of proteoglycans and aggrecan messenger RNA (mRNA) levels were determined. RESULTS: The application of 5 MPa static pressure caused a significant increase in GAG synthesis of 11% (P < 0.05). Column chromatography showed that this increase in GAG synthesis was associated with large proteoglycans. In addition, semiquantitative reverse transcriptase-polymerase chain reaction showed a 4-fold increase in levels of aggrecan mRNA (P < 0.01). CONCLUSION: Hydrostatic pressure in isolation, which does not cause cell deformation, can affect proteoglycan metabolism in chondrocytes cultured in agarose gels, indicating an important role of hydrostatic pressure in the regulation of extracellular matrix turnover in articular cartilage.

Aggrecans↗
PubMed2003

Upregulation of aggrecan and type II collagen mRNA expression in bovine chondrocytes by the application of hydrostatic pressure.

The aim of this study was to investigate the effect of hydrostatic pressure on the expression of messenger ribonucleic acid (mRNA) for specific extracellular matrix proteins in chondrocytes. Chondrocytes obtained from bovine metatarsophalangeal joints were embedded in cylindrical 2% agarose gels. A novel experimental system was used to apply 5 MPa of static hydrostatic pressure to these chondrocytes for 4 hours. The application of hydrostatic pressure caused a significant increase in the level of aggrecan mRNA by almost four fold (p<0.01) as well as a 50% increase in the level of type II collagen mRNA (p<0.05). However, there was no significant change in the level of TIMP-1 mRNA. It was suggested that the application of hydrostatic pressure, in the absence of cell deformation, can bring about changes in the matrix components which may play an important role in the homeostasis and mechanical properties of articular cartilage.

Aggrecans↗