Biomedical subjects

T M Jovin

Publications and source records attributed to T M Jovin.

At least 19 recordsLinked to original sources

PubMed2001

Dynamic interactions of p53 with DNA in solution by time-lapse atomic force microscopy.

Dynamic interactions of the tumor suppressor protein p53 with a DNA fragment containing a p53-specific recognition sequence were directly observed by time-lapse tapping mode atomic force microscopy (AFM) in liquid. The divalent cation Mg(2+) was used to loosely attach both DNA and p53 to a mica surface so they could be imaged by the AFM while interacting with each other. Various interactions of p53 with DNA were observed, including dissociation/re-association, sliding and possibly direct binding to the specific sequence. Two modes of target recognition of p53 were detected: (a) direct binding, and (b) initial non-specific binding with subsequent translocation by one-dimensional diffusion of the protein along the DNA to the specific site.

Aluminum Silicates↗

PubMed2001

Electron and scanning force microscopy studies of alterations in supercoiled DNA tertiary structure.

The configuration of supercoiled DNA (scDNA) was investigated by electron microscopy and scanning force microscopy. Changes in configuration were induced by varying monovalent/divalent salt concentrations and manifested by variation in the number of nodes (crossings of double helical segments). A decrease in the concentration of monovalent cations from 50 mM to approximately 1 mM resulted in a significant change of apparent configuration of negatively supercoiled DNA from a plectonemic form with virtually approximately 15 nodes (the value expected for molecules of approximately 3000 bp) to one or two nodes. This result was in good agreement with values calculated using an elastic rod model of DNA and salt concentration in the range of 5-50 mM. The effect did not depend on the identity of the monovalent cation (Na(+), K(+)) or the nature of the support used for electron microscopy imaging (glow-discharged carbon film, polylysine film). At very low salt concentrations, a single denatured region several hundred base-pairs in length was often detected. Similarly, at low concentrations of divalent cations (Mg(2+), Ca(2+), Zn(2+)), scDNA was apparently relaxed, although the effect was slightly dependent on the nature of the cation. Positively supercoiled DNA behaved in a manner different from that of its negative counterpart when the ion concentration was varied. As expected for these molecules, an increase in salt concentration resulted in an apparent relaxation; however, a decrease in salt concentration also led to an apparent relaxation manifested by a slight decrease in the number of nodes. Scanning force microscopy imaging of negatively scDNA molecules deposited onto a mica surface under various salt conditions also revealed an apparent relaxation of scDNA molecules. However, due to weak interactions with the mica surface in the presence of a mixture of mono/divalent cations, the effect occurred under conditions differing from those used for electron microscopy. We conclude that the observed changes in scDNA configuration are inherent to the DNA structure and do not reflect artifacts arising from the method(s) of sample preparation.

Adsorption↗

PubMed2001

Aromatic amino acids are critical for stability of the bicoid homeodomain.

The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription and translation factor dependent on the unique DNA and RNA binding properties of the homeodomain (HD). We have used circular dichroism and fluorescence spectroscopy to probe the structure and stability of the Bcd-HD, for which a high resolution structure is not yet available. The fluorescence from the single tryptophan residue in the HD (Trp-48) is strongly quenched in the native state but is dramatically enhanced ( approximately 20-fold) upon denaturation. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan fluorescence may be a general phenomenon of HD proteins. We have used site-directed mutagenesis to explore the role of aromatic acids in the structure of the Bcd-HD and to evaluate the proposal that interactions between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native state. We determined that both Trp-48 and Phe-8 in the N-terminal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientation of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.

Amino Acid Substitution↗

PubMed2001

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression.

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.

Animals↗

PubMed2001

Protein-free parallel triple-stranded DNA complex formation.

A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

2,2'-Dipyridyl↗

PubMed2001

A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images.

A programmable array microscope (PAM) incorporates a spatial light modulator (SLM) placed in the primary image plane of a widefield microscope, where it is used to define patterns of illumination and/or detection. We describe the characteristics of a special type of PAM collecting two images simultaneously. The conjugate image (Ic) is formed by light originating from the object plane and returning along the optical path of the illumination light. The non-conjugate image (Inc) receives light from only those regions of the SLM that are not used for illuminating the sample. The dual-signal PAM provides much more time-efficient excitation than the confocal laser scanning microscope (CLSM) and greater utilization of the available emission light. It has superior noise characteristics in comparison to single-sided instruments. The axial responses of the system under a variety of conditions were measured and the behaviour of the novel Inc image characterized. As in systems in which only Ic images are collected (Nipkow-disc microscopes, and previously characterized PAMs), the axial response to thin fluorescent films showed a sharpening of the axial response as the unit cell of the repetitive patterns decreased in size. The dual-signal PAM can be adapted to a wide range of data analysis and collection strategies. We investigated systematically the effects of patterns and unit cell dimensions on the axial response. Sufficiently sparse patterns lead to an Ic image formed by the superposition of the many parallel beams, each of which is equivalent to the single scanning spot of a CLSM. The sectioning capabilities of the system, as given by its axial responses, were similar for a given scan pattern and for processed pseudorandom sequence (PRS) scans with the same size of the unit cell. For the PRS scans, optical sectioning was achieved by a subtraction of an Inc image or, alternatively, a scaled widefield image from the Ic image. Based on the comparative noise levels of the two methods, the non-conjugate subtraction was significantly superior. A point spread function for Ic and Inc was simulated and properties of the optical transfer functions (OTFs) were compared. Simulations of the OTF in non-conjugate imaging did not suffer from the missing cone problem, enabling a high quality deconvolution of the non-conjugate side alone. We also investigated the properties of images obtained by subjecting the Ic and Inc data to a combined maximum likelihood deconvolution.

Animals↗

PubMed2001

Binding of p53 and its core domain to supercoiled DNA.

We have compared the binding of human full-length p53 protein (p53; expressed in bacteria and insects) and its isolated core domain (p53CD, amino acids 94-312; expressed in bacteria) to negatively supercoiled (sc) DNA using gel electrophoresis and immunoblotting. Significant differences were observed; p53CD produced a relatively small and continuous retardation of scDNA, in contrast to the ladder of distinct bands formed by p53 in agarose gels. The ladder produced by full-length protein expressed in bacteria (p53b) was similar to that observed earlier with protein expressed in insect cells (p53i). Competition between scDNAs and their linearized (lin) forms showed a preference for scDNAs by both p53 and p53CD, but the ratios characterizing the distribution of the protein between sc and lin pBluescript DNAs were substantially higher for p53 (sc/lin > 60 in p53b) than for p53CD (sc/lin approximately 4). Strong binding of p53 to scDNA lacking the p53 consensus sequence may represent a new p53-binding mode, which we tentatively denote supercoil-selective (SCS) binding. This binding requires both the C-terminal domain and the core domain. Targets of this binding may include: (a) DNA segments defined both by the nucleotide sequence and local topology, and/or (b) strand crossings and/or bending. The binding preference of p53CD for scDNA may be due to the known nonspecific binding to internal single-stranded regions in scDNA (absent in relaxed DNA molecules) and/or to SCS binding albeit with reduced affinity due to the absence of contributions from other p53 domains.

Baculoviridae↗

PubMed2001

The telomeric dG(GT)4G sequence can adopt a parallel-stranded double helical conformation.

Oligonucleotides 3'-d(GTGTGTGTGG)-L-d(GGTGTGTGTG)-3' (hp-GT) and 3'-d(G4STG4TG4STG4STGG)-L-d(GGTGTGTGTG)-3' (hp-SGT), (L=(CH2CH2O)3), were shown by use of several optical techniques to form a novel parallel-stranded (ps) intramolecular double helix with purine-purine and pyrimidine-pyrimidine base pairing. The rotational relaxation time of hp-GT was similar to that of a 10-bp reference duplex, and the fraction of unpaired bases was determined to be approximately 7%, testifying to the formation of an intramolecular double helical hairpin by the sequence under the given experimental conditions. A quasi-two-state mode of ps-double helix formation was validated, yielding a helix-coil transition enthalpy of -135 +/- 5 kJ/mol. The G x G and T x T (or 4ST x T) base pair configurations and conformational parameters of the double helix were derived with molecular modeling by force field techniques. Repetitive d(GT) sequences are abundant in telomers of different genomes and in the regulatory regions of genes. Thus, the observed conformational potential of the repetitive d(GT) sequence may be of importance in the regulation of cell processes.

Base Pairing↗

PubMed2001

Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation.

Epidermal growth factor receptor (EGFR, erbB1) activation and translocation of the Shc adaptor protein to activated receptors were analyzed at the subcellular level by dual-label immunofluorescence and confocal laser scanning microscopy in conjunction with a new microsphere-based protocol. In the Quantitative Microsphere Recruitment Assay (QMRA) introduced here, epidermal growth factor-coated 1 microm diameter microspheres were distributed over the surface of adherent tissue culture cells expressing the receptor. High-resolution confocal microscopy of a fusion construct of the receptor and the green fluorescent protein expressed in Chinese hamster ovary cells demonstrated that engulfment and internalization of the microspheres occurred rapidly within minutes, and in a receptor activation-dependent manner. In human epidermoid carcinoma A431 cells, receptor activation and Shc translocation persisted over the 20-minute time course of the experiments. However, at the subcellular level the positive correlation of receptor activation and Shc translocation observed at 5-8 minutes dissipated, indicating a time-dependent decoupling of the two events and variation in the kinetics of signal transduction for different subcellular locations.

Animals↗

PubMed2001

Cell fusion experiments reveal distinctly different association characteristics of cell-surface receptors.

The existence of small- and large-scale membrane protein clusters, containing dimers, oligomers and hundreds of proteins, respectively, has become widely accepted. However, it is largely unknown whether the internal structure of these formations is dynamic or static. Cell fusion was used to perturb the distribution of existing membrane protein clusters, and to investigate their mobility and associations. Scanning near-field optical microscopy, confocal and electron microscopy were applied to detect the exchange of proteins between large-scale protein clusters, whereas photobleaching fluorescence energy transfer was used to image the redistribution of existing small-scale membrane protein clusters. Large-scale clusters of major histocompatibility complex (MHC)-I exchanged proteins with each other and with MHC-II clusters. Similarly to MHC-I, large-scale MHC-II clusters were also dynamic. Exchange of components between small-scale protein clusters was not universal: intermixing did not take place in the case of MHC-II homoclusters; however, it was observed for homoclusters of MHC-I and for heteroclusters of MHC-I and MHC-II. These processes required a fluid state of the plasma membrane, and did not depend on endocytosis-mediated recycling of proteins. The redistribution of large-scale MHC-I clusters precedes the intermixing of small-scale clusters of MHC-I indicating a hierarchy in protein association. Investigation of a set of other proteins (alpha subunit of the interleukin 2 receptor, CD48 and transferrin receptor) suggested that a large-scale protein cluster usually exchanges components with the same type of clusters. These results offer new insight into processes requiring time-dependent changes in membrane protein interactions.

Cell Fusion↗

PubMed2000

Parallel-stranded DNA with mixed AT/GC composition: role of trans G.C base pairs in sequence dependent helical stability.

Parallel-stranded (ps) DNAs with mixed AT/GC content comprising G.C pairs in a varying sequence context have been investigated. Oligonucleotides were devised consisting of two 10-nt strands complementary either in a parallel or in an antiparallel orientation and joined via nonnucleotide linkers so as to form 10-bp ps or aps hairpins. A predominance of intramolecular hairpins over intermolecular duplexes was achieved by choice of experimental conditions and verified by fluorescence determinations yielding estimations of rotational relaxation times and fractional base pairing. A multistate mode of ps hairpin melting was revealed by temperature gradient gel electrophoresis (TGGE). The thermal stability of the ps hairpins with mixed AT/GC content depends strongly on the specific sequence in a manner peculiar to the ps double helix. The thermodynamic effects of incorporating trans G.C base pairs into an AT sequence are context-dependent: an isolated G. C base pair destabilizes the duplex whereas a block of > or =2 consecutive G.C base pairs exerts a stabilizing effect. A multistate heterogeneous zipper model for the thermal denaturation of the hairpins was derived and used in a global minimization procedure to compute the thermodynamic parameters of the ps hairpins from experimental melting data. In 0.1 M LiCl at 3 degrees C, the formation of a trans G.C pair in a GG/CC sequence context is approximately 3 kJ mol(-)(1) more favorable than the formation of a trans A.T pair in an AT/TA sequence context. However, GC/AT contacts contribute a substantial unfavorable free energy difference of approximately 2 kJ mol(-)(1). As a consequence, the base composition and fractional distribution of isolated and clustered G.C base pairs determine the overall stability of ps-DNA with mixed AT/GC sequences. Thus, the stability of ps-DNA comprising successive > or =2 G.C base pairs is greater than that of ps-DNA with an alternating AT sequence, whereas increasing the number of AT/GC contacts by isolating G.C base pairs exerts a destabilizing effect on the ps duplex. Molecular modeling of the various helices by force field techniques provides insight into the structural basis for these distinctions.

Base Composition↗

PubMed2000

EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy.

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.

Cytoplasm↗

PubMed2000

Scanning force microscopy of the complexes of p53 core domain with supercoiled DNA.

We used scanning force microscopy to analyse the interaction of the core domain of the tumor suppressor protein p53 (p53CD, amino acid residues 94 to 312), with supercoiled DNA (scDNA) molecules. The complexes were attached to a mica substrate by the divalent cation spreading technique. p53CD bound to supercoiled plasmid pPGM1 bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON) was imaged as a globular complex. Only one such complex was observed with each scDNA molecule. In contrast, binding to supercoiled pBluescript II SK(-) DNA (lacking the consensus sequence) resulted in the appearance of multiple, variable size complexes of various sizes on single DNA molecules. Addition of p53CD to scDNA containing a cruciform-forming (AT)(34) insert resulted in the binding of the protein exclusively at the cruciform. The data presented here suggest that p53CD can form stable specific and non-specific complexes with supercoiled DNA molecules, albeit of variable multimeric organization.

Aluminum Silicates↗

PubMed2000

Photophysics and optical switching in green fluorescent protein mutants.

We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable "one-color" systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in "color" on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.

Green Fluorescent Proteins↗

PubMed2000

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy.

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.

Chromatin↗

PubMed2000

One- and two-photon excited fluorescence lifetimes and anisotropy decays of green fluorescent proteins.

We have used one- (OPE) and two-photon (TPE) excitation with time-correlated single-photon counting techniques to determine time-resolved fluorescence intensity and anisotropy decays of the wild-type Green Fluorescent Protein (GFP) and two red-shifted mutants, S65T-GFP and RSGFP. WT-GFP and S65T-GFP exhibited a predominant approximately 3 ns monoexponential fluorescence decay, whereas for RSGFP the main lifetimes were approximately 1.1 ns (main component) and approximately 3.3 ns. The anisotropy decay of WT-GFP and S65T-GFP was also monoexponential (global rotational correlation time of 16 +/- 1 ns). The approximately 1.1 ns lifetime of RSGFP was associated with a faster rotational depolarization, evaluated as an additional approximately 13 ns component. This feature we attribute tentatively to a greater rotational freedom of the anionic chromophore. With OPE, the initial anisotropy was close to the theoretical limit of 0.4; with TPE it was higher, approaching the TPE theoretical limit of 0.57 for the colinear case. The measured power dependence of the fluorescence signals provided direct evidence for TPE. The general independence of fluorescence decay times, rotation correlation times, and steady-state emission spectra on the excitation mode indicates that the fluorescence originated from the same distinct excited singlet states (A*, I*, B*). However, we observed a relative enhancement of blue fluorescence peaked at approximately 440 nm for TPE compared to OPE, indicating different relative excitation efficiencies. We infer that the two lifetimes of RSGFP represent the deactivation of two substates of the deprotonated intermediate (I*), distinguished by their origin (i.e., from A* or B*) and by nonradiative decay rates reflecting different internal environments of the excited-state chromophore.

Amino Acid Substitution↗

PubMed2000

Three-dimensional spectral imaging by hadamard transform spectroscopy in a programmable array microscope.

We report the acquisition and deconvolution of three-dimensional spectrally resolved images in a programmable array microscope implementing a Hadamard transform fluorescence spectroscopy system with adjustable spectral resolution. A stack of 16 two-dimensional spectral images was collected at 400 nm intervals along the optical axis. The specimen consisted of a polytene chromosome spread from Drosophila melanogaster doubly labelled for the Polyhomeotic protein by indirect immunofluorescence labelling with Alexa594 and for DNA with YOYO-1. The resulting four-dimensional data set consisted of the xyz spatial dimensions (898 x 255 x 16) with a 26-point spectrum at each spatial location. The total exposure time to the sample was 34 min. The system requires the acquisition of multiple images, and thus works best with fluorophores that are resistant to photobleaching. Image deconvolution reduced the amount of out-of-focus blur by up to a factor of 8, resulting in a dramatic improvement in the visualization of the chromosome backbone and localization of the specific Polyhomeotic domains.

Animals↗

PubMed2000

HMG1 protein stimulates DNA end joining by promoting association of DNA molecules via their ends.

High mobility group (HMG) 1 protein is a highly abundant and an evolutionarily conserved chromosomal protein with two homologous DNA-binding domains (HMG boxes), A and B, attached by a short basic region to an acidic C-terminal tail. The protein has been implicated in a number of fundamental biological processes including DNA replication, transcription, recombination and repair. We demonstrate that HMG1 is able to enhance cohesive-end and blunt-end DNA ligation by T4 DNA ligase via its B domain. The C-terminal flanking sequence of the B domain (seven basic residues out of approximately 18) and a number of conserved amino-acid residues within the HMG box (mainly basic or hydrophobic) are required for efficient stimulation of ligation. Pull-down assays, electron and scanning force microscopy revealed that HMG1 can associate two DNA molecules via their ends even in the absence of complementary overhangs. We propose that HMG1 protein may be involved in the rejoining of DNA breaks by different DNA ligases due to its ability to bring DNA duplexes and their termini into a close proximity while leaving the ends accessible for ligation.

Amino Acid Sequence↗