Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage lambda vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable lambda sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.     

Isolation and characterization of pock-forming plasmids for Streptomyces griseus from soil actinomycetes

Thirty independent actinomycetes strains carrying plasmids were isolated from soil. These plasmids were purified as cccDNA by CsCl-EtBr equilibrium density-gradient centrifugation. Plasmids that induce "pocks", namely formation of circular zones of sporulation-inhibition, were selected by protoplast transformation of streptomycin-producing strain, Streptomyces griseus ATCC10137. Six pock-forming plasmids, pOA7, pOA11, pOA15, pOA23, pOA29 and pOA30, were obtained, and their cleavage maps, transformation frequencies, and copy numbers, as well as their stability, are described.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Presynaptic dense bars at neuromuscular synapses of the lobster,Homarus americanus

Summary The threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.Supported by Muscular Dystrophy Association of Canada and NSERCC. Generous use of laboratory facilities at Woods Hole was provided by the late Fred Lang     

Fluorescence microscopy of the endomembrane system of living plant cells

Abstract The fluorochrome Auramine O has been evaluated as a fluorescent probe for components of the endomembrane system of living plant cells. At 0.001% w/v the compound did not inhibit seedling growth or cytoplasmic streaming but stained the nuclear envelope, endoplasmic reticulum and Golgi apparatus. The three-dimensional, structural interrelationships of these organelles in living tissues could be resolved after minimal tissue preparation. The method is also a valuable control treatment for use in conjunction with electron microscope fixation procedures. It provides a rapid means of examining dynamic changes in the endomembrane system associated with cell development and differentiation and could have application in monitoring the effects of applied physiological or chemical stress.     

Stromatolite-dominated microbialites at the Permian–Triassic boundary of the Xikou section on South Qinling Block,China

Permian–Triassic boundary microbialites (PTBMs) are organosedimentary carbonates formed immediately after the end-Permian mass extinction. All those reported PTBMs constrained by convincing conodont biozones are present stratigraphycally not higher than the Hindeodus parvus zone and most of them are dominated by thrombolites. This paper provides the first record of a brief, but spectacular development of stromatolite-dominated PTBMs within the basal Isarcicella isarcica conodont zone of the earliest Triassic from the Xikou section of South Qinling Block that was at the margin of the North China Block during the Permian–Triassic transition and was geographically separated from the major occurrence of post-extinction microbialites in the South China Block. This stromatolite cap overlies a 3.7-m-thick oolitic limestone and is composed of a lower 0.2-m-thick bed and an upper 0.5-m-thick bed, separated by a 0.2-m-thick greyish green siliciclastic mudstone. These two stromatolite beds mainly consist of columnar stromatolites with subordinate domal stromatolites. The intercolumn and interstitial spaces within the stromatolites are filled with oolitic grainstones. At the microscopic scale, laminoid structures in stromatolites comprise wavy, millimetric-domical and tangled laminae. The increased grain and fossil contents and/or bioturbation in the domical and tangled laminae indicate that the formation of these laminae is likely related to an increase in the populations and the disruptions by benthic metazoans, as well as an influx of sediment grains. The δ¹³C_carb values fluctuate between 2‰ and 3‰ in the uppermost Permian strata; a distinct negative shift of 1.9‰ occurs at the topmost oolitic grainstone, just below the lower stromatolite bed, and the lowest value of −0.1‰ is located at the base of the upper stromatolite bed. The stratigraphic succession from stromatolites to thrombolites of the PTBMs may represent a transgressive succession and/or a transient ecosystem recovery immediately after the end-Permian mass extinction. The thrombolites-dominated PTBMs mainly developed in near-equator shallow marine geographic locations, and stromatolite-dominated PTBMs mainly developed at higher latitude settings, which probably indicates that a relatively lower diversity and abundance of marine benthic metazoans existed at higher latitudes after the end-Permian mass extinction.     

Organic-walled microfossils from Cambrian Stage IV in the Jiaobang section,eastern Guizhou,China

The lower Cambrian succession in the Jiaobang section, Jianhe County, eastern Guizhou, China, includes, in ascending order, the Bianmachong, Balang, and Tsinghsutung formations, with a total thickness of about 645 m. Twenty-six morphological genera (including one new genus) are identified from the Balang and the underlying Bianmachong formations, many of which are common and widely distributed. Six acritarch assemblages are discerned in the Balang Formation. They are, in ascending order, the Adara alea‒Skiagia ornata, the Acrum radiale‒Pterospermella velata, the Comasphaeridium molliculum‒Solisphaeridium baltoscandium, the Corrugasphaera perfecta n. sp.‒Pterospermella vinctusa n. sp., the Acrum novum‒Heliosphaeridium oligum, and the Acrum membranosum‒Adarve diafanum acritarch assemblages. An obvious change of organic-walled microfossil assemblages occurred in the interval between 84 m and 98 m from the bottom of the Balang Formation which roughly corresponds to the boundary between the Oryctacarella duyunensis trilobite Zone and the overlying Arthricocephalus chauveaui trilobite Zone. In addition, organic-walled microfossils are scarce in about 24 m thick from the bottom of the Balang Formation. One new genus and five new species including Plagasphaera balangensis n. gen. n. sp., Asteridium tubulus n. sp., Cymatiosphaera spina n. sp., Corrugasphaera perfecta n. sp., and Pterospermella vinctusa n. sp. are described.     

A Method for Making Aceto-Carmin Smears Permanent

Anthers are collected and placed in a solution of 1 part acetic acid to 3 parts of absolute alcohol. The contents of the anther are squeezed out on a slide in a drop of Belling's iron-aceto-carmin solution and a cover glass placed over the drop. Care should be taken to remove all anther walls and flower parts. Heat the slide over an alcohol flame for a second, repeating 4 or 5 times. Place the slide in a petri dish filled with a 10% solution of acetic acid. When the cover glass has risen away from the slide gently remove the cover glass and place in a Coplin jar containing equal parts of alcohol and acetic acid. Likewise, place the slide in this solution. Run both cover and slide thru the following solutions: 1 part acetic acid to 3 parts absolute alcohol, 1 part acetic acid to 9 parts absolute alcohol, absolute alcohol and finally equal parts of absolute alcohol and xylol. Recombine the cover and slide in xylol-balsam directly from this solution.     

A focus on fixation

Three fixation issues related to immunostaining are discussed here: 1) Generally, a tissue block is fixed, then embedded and sectioned (pre-fixation). The type of fixative applied, crosslinking or coagulating, has an impact on selecting an epitope retrieval method. Individual antigens have a fixation–retrieval characteristic. 2) A long fixation time, especially with crosslinking fixatives, may compromise the result of immunostaining. This negative effect varies among different antigens and can be partially restored by applying a more sensitive/efficient detection system such as tyramide amplification. 3) Sections cut from a fresh frozen tissue block usually are acetone fixed (post-fixation). This was accepted as the “gold standard” for a long time. Post-fixation, however, may have serious consequences for preservation of small peptides leaking from the cut open cells, whereas this is not the case with pre-fixed intact cells. Consequently, the concept of an acetone post-fixed cryostat tissue section as “gold standard” no longer exists and a more appropriate use of the terms immunohistochemistry and immunocytochemistry therefore seems justified. For many antibodies, it is not known whether a formalin fixed, paraffin embedded tissue specimen is appropriate. Suggestions are made for creating a positive control cell block for testing such antibodies.