Study of plasmid replication in Escherichia coli with a combination of 2D gel electrophoresis and electron microscopy.

We studied theta-mode DNA replication in p15A-based Escherichia coli plasmids by analyzing their replication intermediates using a combination of neutral agarose 2D gel electrophoresis and electron microscopy. Our analysis: (1) confirms the original assignment of various features of the 2D gel pattern; (2) shows that while one replication fork progresses around the plasmid DNA, the other is immobile, as if the replication were unidirectional; and (3) reveals that termination often occurs at a location away from the replication origin, suggesting that the replication of our plasmids is, in fact, bidirectional, the two forks being active at different times.

Structural analysis of membrane-bound retrovirus capsid proteins.

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles.

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double-strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD-promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD-chi site interactions in vivo we used phage lambda's terminase to introduce a site-specific double-strand break at lambda's cos site inserted into a plasmid.

Masking generates contiguous segments of metal-coated and bare DNA for scanning tunneling microscope imaging.

To date, no microscopic methods are available to confirm scanning tunneling microscope (STM) images of DNA. The difficulties encountered in repeating these images may be attributed to inadequate distribution of molecules on the substrate, poor adhesion to the substrate, or the low conductivity of the molecules. However, these factors are difficult to assess in an STM experiment where they may act simultaneously.

Deposition and imaging of metal-coated biomolecules with the STM.

We have applied a simple and reliable procedure for imaging biomolecules with the scanning tunneling microscope (STM). The biomolecules are adsorbed on glow-discharged mica, then coated with a thin film of platinum-carbon. We have tested this method with linear and circular (plasmid) DNA molecules.

Assembly of Octopus dofleini hemocyanin. A study of the kinetics by sedimentation, light scattering and electron microscopy.

The kinetics of association of Octopus dofleini hemocyanin subunits to form the native decameric molecule have been studied with a combination of sedimentation, light scattering and electron microscopy. The reaction, initiated by addition of magnesium, is relatively slow, requiring hours to reach completion, with monomer and decamer as predominant molecular species throughout. Analysis of the light-scattering data, including stopped-flow studies, reveals an initial lag period in the reaction, followed by a second-order process that is rate limiting.

Arrangement of subunits and domains within the Octopus dofleini hemocyanin molecule.

Native Octopus dofleini hemocyanin appears as a hollow cylinder in the electron microscope. It is composed of 10 polypeptide subunits, each folded into seven globular oxygen-binding domains. The native structure reassociates spontaneously from subunits in the presence of Mg2+ ions.

Repeat-induced G-C to A-T mutations in Neurospora.

In the Neurospora genome duplicate sequences are detected and altered in the sexual phase. Both copies of duplicate genes are inactivated at high frequency, whether or not they are linked. Restriction sites change, and affected sequences typically become heavily methylated.

Isolation and reactivation of highly-coupled newt lung cilia.

The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5-5.0 cm in length.

Factors guiding optic fibers in developing Xenopus retina.

We have characterized, by electron microscopy, the growth of pioneering axons from the retina into the visual pathway during early development of Xenopus laevis. The subsequent development of following fibers from the growing retinal margin as they accumulated in the ganglion cell fiber layer (GCFL) of the retina was also studied. Extracellular channels bordered by neuroepithelial cells appear in the developing retina in a dorsal to ventral gradient before any pioneering axons are seen.

Cell wall and lipid composition of Isosphaera pallida, a budding eubacterium from hot springs.

Isosphaera pallida is an unusual gliding, budding eubacterium recently isolated from North American hot springs. Electron micrographs of ultrathin sections revealed a cell wall atypical of eubacteria: two electrondense layers separated by an electron-transparent layer, with no evident peptidoglycan layer. Growth was not inhibited by penicillin.

Anatomy of the posterior lateral line system in young larvae of the zebrafish.

We studied the anatomy of neuromasts, afferent sensory neurons, and efferent neurons of the midbody branch of the posterior lateral line in larvae of the zebrafish (Brachydanio rerio), 5 days after fertilization. This simple sensory system consists of ten or 11 neuromasts, 15-20 sensory neurons, and about nine efferent neurons. The neuromasts are typical free neuromasts and both afferent and efferent synapses are present on hair cells within them.

T reticular interneurons: a class of serially repeating cells in the zebrafish hindbrain.

We describe a class of reticular neurons, named T interneurons after the branching pattern of their axons, in young larvae of the zebrafish Brachydanio rerio. The cells were identified by filling them with HRP from application sites within the CNS. A serially repeating set of about ten such neurons is present in a longitudinal column on each side of the caudal hindbrain.

Functional development in the Mauthner cell system of embryos and larvae of the zebra fish.

In the embryonic zebra fish as early as 40 hr after fertilization, the Mauthner cells (M-cells) initiate an escape response, elicited by tactile-vibrational stimulation. The initial part of this behavior is similar to the acoustic startle reflex seen during the larval stage which begins at 96 hr. The embryonic response is directional and is followed by a series of strong tail flexures which are more pronounced than those during swimming.

The quaternary structure of a molluscan (Helisoma trivolvis) extracellular hemoglobin.

The hemoglobin (erythrocruorin) of the planorbid mollusc Helisoma trivolvis has a molecular weight of 1.7-10(6) and a sedimentation coefficient (s0 20, w) of 33.8 S at pH 7.

The fine structure of the male Drosophila genital disk during late larval and early pupal development.

The fine structure of male genital disks of Drosophila between the late second larval instar and early pupal stage is described. Two major developmental changes were observed. One, the incidence of rough endoplasmic reticulum increases around the time of puparium formation.