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Biomedical subjects

M Seville

Publications and source records attributed to M Seville.

10 recordsLinked to original sources
PubMed2001

A whole new way of looking at things: the use of Dark Reader technology to detect fluorophors.

The Dark Reader optical system (Clare Chemical Research, Denver, CO, USA) uses relatively low intensity broad-band visible blue light in combination with broad-band optical filters to detect fluorescence with a level of sensitivity that often surpasses that of UV transilluminators and can rival that of laser-based scanners. Applications of DR (Clare Chemical Research) devices include the detection of DNA and SYBR-stained protein samples following, and also during, electrophoresis. Unlike laser-based imaging systems, the fluorescence is directly visible to the user as well as being fully compatible with charge-coupled device (CCD) and Polaroid camera-based detection and imaging. Additionally, the DR optical system functions well in multicolor fluorophor environments. Because the Dark Reader does not emit any UV light, the extent of DNA damage incurred when visualizing DNA samples is drastically reduced compared to the damage produced by a UV device and this can have a significant benefit on downstream cloning protocols. Furthermore, dye photobleaching is minimal, extending the length of time that a fluorescent sample is visible. The inherent flexibility of the DR optical system allows many different configurations of the Dark Reader to be constructed such as transilluminators, hand lamps and integrated transilluminator-electrophoresis units.

DNA↗
PubMed1997

A DNA polymerase III holoenzyme-like subassembly from an extreme thermophilic eubacterium.

We have purified a novel DNA polymerase from Thermus thermophilus. This was enabled by use of general gap filling assays to monitor polymerase activity and cross-reactive monoclonal antibodies against the alpha catalytic subunit of E. coli DNA polymerase III holoenzyme to distinguish a novel polymerase from the well characterized DNA polymerase I-like Thermus thermophilus DNA polymerase. Two proteins migrating with the polymerase after three chromatographic steps were isolated and subjected to partial amino acid sequencing. The amino termini of both were homologous to the two products of the E. coli dnaX gene, the gamma and tau subunits of the DNA polymerase III holoenzyme. Using this information and sequences conserved among dnaX-like genes, we isolated a gene fragment by PCR and used it as a probe to isolate the full length Thermus thermophilus dnaX gene. The deduced amino acid sequence is highly homologous to the DnaX proteins of other bacteria. Examination of the sequence permitted identification of a frameshift site similar to the one used in E. coli to direct the synthesis of the shorter gamma DnaX-gene product. Based on this information, we conclude that a conventional replicase exists in extreme thermophilic eubacteria. The general biological and practical technological implications of this finding are discussed.

Amino Acid Sequence↗
PubMed1996

Fluorometric assay for DNA polymerases and reverse transcriptase.

We report a quick, easy and inexpensive fluorometric assay that measures the activity of replication enzymes using Pico-GreenTM. The systems tested include replication of the natural template M13 Gori by E. coli DNA polymerase III holoenzyme and the replication of a synthetic homopolymer by human immunodeficiency virus reverse transcriptase. A direct comparison of the fluorometric assay with the conventional isotopic assay shows that the fluorometric assay accurately reflects the extent of replication. By performing the assay reactions directly in 96-well plates and using a fluorescence plate reader to determine the extent of reaction, the time required to measure replication activities is significantly shortened.

DNA-Directed DNA Polymerase↗
PubMed1994

Movement of the F40 domain of flagellin during the morphological transition of bacterial flagella.

Flagella from Salmonella typhimurium were labeled with various amounts of fluorescein isothiocyanate. The site of labeling was identified as being predominantly in the exterior F40 domain. The fluorescence intensity decreased as the fluorescein density on the flagella increased, indicating self energy transfer between fluoresceins. The fluorescence of modified flagella was measured during the normal-to-curly morphological transition induced by alkaline pH. The morphological transition itself was simultaneously monitored by dark-field microscopy. Concomitant with the transition was a 25% increase in fluorescence for flagella heavily labeled with fluorescein. This was shown to be due to a decrease in the efficiency of energy transfer between fluoresceins on proximal flagellin subunits, implying that the F40 domains undergo relative movement apart during the morphological transition. Closer inspection of the domain movement and morphological transition as a function of pH reveals that the two processes are not exactly concomitant. This indicates the existence of intermediates during the transition. The fluorescence technique, outlined here, provides a means of directly monitoring an organizational 'switch' in the flagellin subunits during the actual morphological transition of flagella.

Flagella↗
PubMed1994

Significant improvement to the catalytic properties of aspartate aminotransferase: role of hydrophobic and charged residues in the substrate binding pocket.

The substrate specificity of tyrosine aminotransferase (eTAT) from Escherichia coli has been tested by transferring the critically different residues Leu39, Glu141, and Arg293 into equivalent positions of aspartate aminotransferase (eAAT). These residues are not directly involved in the catalytic process. The single mutant eAAT V39L possesses greater values of kcat/KM not only for tyrosine but also for aspartate and glutamate. In contrast, the double mutant eAAT P141E,A293R and also the triple mutant eAAT V39L,P141E,A293R exhibit smaller changes of kcat/KM. The converse mutants of tyrosine aminotransferase, in which critical residues of eAAT (Val39) and of mitochondrial AAT (Ala39, Val37) were transferred into equivalent positions of eTAT, exhibited generally decreased values of kcat/KM for both dicarboxylic and aromatic substrates. On the basis of the known structures of eAAT and eAAT V39L as well as of a refined model of eTAT, these results indicate that the different substrate specificities of eAAT and eTAT are due to multiple side chain differences and minor rearrangements of the backbone. The generally improved catalytic efficiency of the mutant eAAT V39L appears to be due to an indirect effect, namely, the facilitated closure of the active site upon substrate binding.

Amino Acid Sequence↗
PubMed1993

The effect of sugars on the morphology of the bacterial flagellum.

Using dark-field microscopy, we have found that certain sugars cause the normal-to-curly helical transition of bacterial flagella. Titration of flagella isolated from Salmonella typhimurium with 16 different carbohydrates showed that: (i) only certain sugars cause the transition. There is no obvious relationship between the simple physico-chemical properties of the sugar and whether the sugar causes the transition or not; (ii) the efficacies of sugars that do cause the transition differ markedly. For these sugars there is a relationship between efficacy and molecular size. These results suggest that the specific, though weak, binding of sugars to sites on flagella cause the morphological transition.

Carbohydrate Conformation↗
PubMed1988

Modeling the three-dimensional structures of bacterial aminotransferases.

The refined crystallographic structure of the "closed" conformation of chicken mitochondrial aspartate aminotransferase has been used as a template for the construction of models of the two Escherichia coli aminotransferases encoded by the tyrB and aspC genes. The main results are as follows: (1) Only minor changes are required in the coordinates of the backbone atoms to accommodate the large number of substituted side chains. (2) All deletions and insertions required to allow maximum primary sequence alignment are on the solvent-accessible surface. (3) Charged residues are all located on the surface, in contact with solvent, except for certain conserved active site residues. (4) The close packing within the hydrophobic core is maintained. (5) The interactions between the subunits are maintained. (6) Modeling of tyrosine as an external aldimine into the active sites points to several residues that could be involved in determining the substrate specificities of these aminotransferases.

Binding Sites↗
PubMed1984

A study of the interaction between bovine cardiac-muscle cyclic AMP-dependent protein kinase and cyclic AMP using fluorescence-polarization spectroscopy.

The C-subunit of type II cyclic AMP-dependent protein kinase from bovine heart was labelled with the fluorophore fluorescamine (FAM). The association of the dye-labelled subunit (CFAM) with the R-subunit isolated from the same source was monitored by fluorescence polarization spectroscopy. The stoichiometry of C to R in the final complex was close to 1:1. The affinity of the two subunits could be described by a dissociation constant in the nanomolar range. Holoenzyme (formed from CFAM and R) was titrated with cyclic AMP, and the changes in fluorescence anisotropy, due to dissociation of the holoenzyme, recorded. The titration curves were analysed in terms of a model which required computer simulation. Cyclic AMP-induced dissociation proceeds via one or more ternary complexes, and all four cyclic AMP-binding sites on the R-dimer are accessible in the holoenzyme. The dissociation constants describing the release of the C-subunits from the two ternary complexes containing four cyclic AMP molecules were both approx. 9 microM. The binding of two cyclic AMP molecules to protein kinase is necessary and sufficient to cause the dissociation of both C-subunits. The state of association at 'in vivo' concentrations of protein and cyclic AMP is discussed.

Animals↗
PubMed1984

The preparation of cyclic nucleotide-depleted regulatory subunits from type II adenosine 3':5'-monophosphate-dependent protein kinase by a nondenaturing method.

A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.

Animals↗