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. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinct cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.

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. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi-less DNA. Increased survival of chi-containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi-containing DNA molecules leads to RecA-dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double-strand break repair.

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. A method to isolate these factors involves partly masking the deposited molecules before coating them with a conductive film to produce adjacent segments of coated and bare DNA after the mask is removed. The coated DNA segments are conductive and mechanically stable to allow easy identification of DNA by the STM. Furthermore, the path of a molecule can be traced from a coated to an uncoated region to test STM imaging of bare DNA. Masked preparations of DNA deposited on platinum/carbon-coated mica and highly oriented pyrolytic graphite were examined with a tunneling current 1000 times lower than the usual nanoamps. The tip apparently displaces molecules adsorbed to graphite to preclude imaging whereas more stably bound DNA on platinum/carbon-coated mica appears in reversed contrast.

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. The contrast and resolution of the STM images are comparable to electron micrographs of the same molecules when shadowed. Though the present lateral resolution (5-6 nm) is limited by the grain size of the conductive film, some details like supercoiled regions in the DNA are resolved. This method is interesting for two reasons. First, as an alternative technique for imaging biomolecules. Second, for use as a control in STM studies of bare biomolecules.

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. The lag period depends on both protein and magnesium ion concentration. Electron microscope studies reveal intermediates in the process, and support a model of assembly in which nucleation begins at the dimer level. Theoretical models for the process are compared.

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. We have selectively removed the C-terminal domain and purified the resulting six-domain subunits. Although these six-domain subunits do not associate efficiently at pH 7.2, they undergo nearly complete reassociation at pH 8.0. The resulting molecule looks like the native cylindrical whole molecule but lacks the usual fivefold protrusions into the central cavity. Partially reassociated mixtures show dimers of the subunit that have a characteristic parallelogram shape when lying flat on the electron microscope grid, and a "boat" form in side view. Removal of the C-terminal domain from monomers results in the removal of two characteristically placed domains in the dimers. These observations allow the development of a model for the arrangement of the subunits within the whole molecule. The model predicts exactly the views seen in the electron microscope of both whole molecule and dimeric intermediates.

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. To characterize the alterations of the DNA, duplicated sequences were isolated before and after one or more sexual cycles. DNA sequencing and heteroduplex analyses demonstrated that the process (termed RIP) produces exclusively G-C to A-T mutations. Changes occur principally at sites where adenine is 3' of the changed cytosine. A sequence duplicated at a distant site in the genome lost approximately 10 percent of its G-C pairs in one passage through a cross. A closely linked duplication of the same sequence that was passed twice through a cross lost about half of its G-C pairs. The results suggest a mechanism for the RIP process.

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. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12-13 micron in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X-100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21 degrees C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.

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. Pioneering axons are subsequently observed in these channels, usually surrounded by neuroepithelial cell processes. Ruthenium red treatment of embryonic retinas reveals extracellular matrix (ECM) within these retinal channels, while extracellular spaces in the proximal optic stalk, just beyond the optic disc, lack this material. ECM is also seen in optic tectum wherever ingrowing retinal and nonretinal axons are found. The channels and the ECM contained within them may provide guidance cues for pioneering retinal axons. The early association of pioneering retinal axons with neuroepithelial cell processes (putative glia) appears to be important in further development of the GCFL. The so-called following fibers of ganglion cells, arising later in development, fasciculate with pioneer axons in extracellular spaces and form fiber bundles of the GCFL on top of the layer of glial cell endfeet. It is not clear whether pioneering axons, glial cell surfaces, or both serve as guidance cues for following fiber migration.

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. Cell walls were isolated from sheared cells by velocity sedimentation. The rigid-layer fraction, prepared from cell walls by treatment with boiling 10% sodium dodecyl sulfate, was hydrolyzed and chemically analyzed for muramic acid. This essential component of peptidoglycan was absent. Amino acid analysis demonstrated a proteinaceous wall structure. Pitlike surface structures seen in negatively stained whole cells and thin sections were correlated with periodically spaced perforations of the rigid sacculus. An analysis of the lipid composition of I. pallida revealed typical ester-linked lipids with unbranched fatty acids, in contrast to the isoprenyl ether-linked lipids of archaebacteria, which also have proteinaceous cell walls. Capnoids, unusual sulfonolipids which are present in gliding bacteria of the Cytophaga-Flexibacter group, were absent.

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. The T axons project across the midline, and branches course both rostrally and caudally within the medial longitudinal fascicle (mlf). The cells receive synaptic input from the Mauthner neurons, from unidentified axons in the mlf, and perhaps from trigeminal sensory fibers. They project to cranial and pectoral motor nuclei. T interneurons appear to be homologous to giant fiber neurons in the hatchetfish and to some of the cranial relay neurons in the goldfish. We discuss a possible functional role and comparative implications of their distribution in the hindbrain.

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. The sensory neurons project into a single longitudinal column of neuropil in the hindbrain. The sensory terminals appear by light microscopy to contact the dorsolateral dendrite of the ipsilateral Mauthner cell. Three types of efferent neurons are present; two types in the hindbrain and one type in the diencephalon. We provide several lines of evidence that demonstrate that these central neurons are efferent to the lateral line. We conclude from this morphology that the larval system includes all of the components of the adult system and is probably functional at this early stage. We also found that larvae have all of the efferent neurons found in adult zebrafish, while the number of neuromasts and sensory neurons will increase during subsequent development.

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. In the embryo the M-cell fired at the beginning of the response and rarely fired again during subsequent contractions; in our experiments the M-cell did not mediate iterative movements of the tail. The M-cell system is probably involved in evoked hatching behavior, as the tactile response is sufficient to rupture the egg membrane and allow the animal to escape. The M-cell sometimes fired spontaneously, which suggests that it might function also in spontaneous hatching behavior which occurs in the absence of phasic stimulation. At 48 hr the M-cell has morphologically mature synapses on its soma and dendrites, but its cytoplasm is relatively undifferentiated; it has few oriented neurofilaments and no distinct axon hillock. During these stages the extracellular M-spike is longer in duration and smaller in amplitude than at later times when the cell is more mature morphologically. Our data suggest that long-term inhibitory control of the M-cell system begins to function at about the time of hatching. At this time the cell is morphologically mature and is richly supplied with synaptic endings over its soma and dendrites.

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 7.0. At pH 2.0, the pigment consists of 32 S and 13 S material. The hemoglobin exists as a 350 000 molecular weight submultiple in 6 M guanidine and can be further dissociated into a 175-200 000 dalton polypeptide in 6M guanidine, 0.1 M 2-mercaptoethanol or by sodium dodecyl sulfate gel electrophoresis of globin, performic acid oxidized globin or carboxymethylated globin. Electron microscope observations show a ten-membered ring structure measuring 200 A in diameter. It is proposed that Helisoma hemoglobin consists of a 1.7-10(6) dalton circular assembly of ten 175-200 000 dalton polypeptide chains. The amino acid composition of the pigment is reported. The hemoglobin contains one heme per 18-19 000 g protein. Limited proteolysis of the intact pigment shows 60 000, 40 000 and 17 000-18 500 dalton components when analyzed by sodium dodecylsulfate gel electrophoresis. It is likely that the 175-200 000 dalton polypeptide consists of a linear arrangement of 8-12 heme-containing domains, each domain having a molecular weight of 18-19 000.