Construction of a human chromosome 3 specific NotI linking library using a novel cloning procedure.

Two new diphasmid vectors (lambda SK17 and SK22) and a novel procedure to construct linking libraries are described. A partial filling-in reaction provides counter-selection against false linking clones in the library, and obviates the need for supF selection. The diphasmid vectors, in combination with the novel selection procedure, have been used to construct a chromosome 3 specific NotI linking library from a human chromosome 3/mouse microcell hybrid cell line. The application of the new vectors and the strong biochemical and biological selections resulted in a library of 60,000 NotI linking clones. As practically all of them are real NotI linking clones (no false recombinants) the library represents approximately 3,000 human recombinants (equal to 10-15 genomic equivalents of chromosome 3). Previously published methods for construction of linking libraries are compared with the procedure described in the present paper. The advantages of the new vectors and the novel protocol are discussed.     

Light affects the structure of Chlamydomonas chloroplast chromosomes.

We have analyzed changes in the structure of chloroplast chromosomes in response to light in growing Chlamydomonas cells using a crosslinking assay based on the intercalation of HMT (4'-hydroxymethyl-4,5',8-trimethylpsoralen) into DNA. Our results show that the structure of chloroplast chromosomes in at least three widely separated regions is different in light-grown vs. dark-grown cells. Structural changes in chloroplast chromosomes occur within 3 hrs after exposure to light or darkness, respectively. The response to light is not inhibited by atrazine and can be elicited by dim blue light incapable of evolving O2, indicating that it does not require photosynthesis. Inhibition of cytoplasmic protein synthesis with cycloheximide prevents this response to light, indicating that it depends, at least in part, on proteins imported from the cytoplasm.     

Efficient site directed in vitro mutagenesis using ampicillin selection.

A novel plasmid vector pSELECT-1 is described which can be used for highly efficient site-directed in vitro mutagenesis. The mutagenesis method is based on the use of single-stranded DNA and two primers, one mutagenic primer and a second correction primer which corrects a defect in the ampicillin resistance gene on the vector and reverts the vector to ampicillin resistance. Using T4 DNA polymerase and T4 DNA ligase the two primers are physically linked on the template. The non-mutant DNA strand is selected against by growth in the presence of ampicillin. In tests of the vector, highly efficient (60-90%) mutagenesis was obtained.     

Control of respiratory motor pattern by sensory neurons in spinal cord of lamprey

Summary Extracellular stimulation over the dorsal funiculus in the spinal cord of lampreys was found to selectively activate prolonged episodes of fictive arousal respiration (Figs. 1, 3). The induced episodes showed comparable increases in cycle frequency and motoneuron burst duration to the spontaneous arousal pattern observed in isolated brain preparations (Fig. 2). Intracellular stimulation of primary sensory neurons with axons in the dorsal funiculus, called dorsal cells, also elicited the arousal pattern (Fig. 4). Mechanoreceptive dorsal cells respond to cutaneous stimulation. When mechanical stimuli were applied to the skin of intact lampreys (Fig. 6) or to lampreys with ipsilateral vagotomy, arousal respiration was induced (Figs. 7, 8). Bilateral, but not unilateral, trigeminal lesion blocked dorsal cell induction of the arousal response (Fig. 5). Spontaneous arousal respiration was recorded from intact, unrestrained lampreys (Fig. 9). These results suggest that fictive arousal respiration is the in vitro correlate of natural arousal respiration in lampreys, and that one mechanism leading to arousal respiration may be the activity of sensory dorsal cells. A model for respiratory motor pattern switching in lamprey is proposed. The model suggests that the normal and arousal patterns are produced by separately engaging rostral or caudal pattern generators in the medulla, rather than by modifying one pattern generator (Fig. 10).     

Evolutionary ethics,Darwinian ethics and ethical naturalism

The relevance of evolutionary theory to ethics goes back to Darwin but until recently discussion employed evolutionary theory to justify ethical, social and political positions. Recently, evolutionary theory has been used to explain the existence of moral systems and moral propensities and, thereby, to provide a naturalistic basis for ethics. I argue that this approach has advanced our understanding of the basis of moral systems and moral propensities but does not as yet adequately incorporate the role of cognition in its account. Cognition has the effect of decoupling to some extent — though, of course, far from fully — human moral systems from their evolutionary origins. In an adequate account, evolutionary theory will play a crucial role but so also will our evolved cognitive abilities.     

Tissue architectural features for the grading of prostatic carcinoma

In research for the development of a computer-aided workstation for the objective grading of prostatic carcinoma, tissue architectural (histometric) features were analyzed in ten cases each of well-differentiated, moderately differentiated and poorly differentiated carcinoma (as subjectively graded by the consensus of a panel of experts). Sections were cut at 4 microns, stained by the Feulgen reaction and digitized by two different video-based photometric systems. Some images were interactively segmented, considering the histometric clues to be studied; others were automatically segmented by an expert system-guided technique. The latter procedure produced good results, with over 90% of the nuclei judged to be correctly segmented in 64% of the fields studied and over 80% in another 24% of the fields. While the number of nuclei per field provided some separation of well-differentiated from other lesions, the number of nuclei per gland distinguished between well-differentiated and moderately differentiated lesions. Simplicial decomposition of the images also provided a measure of the degree of differentiation, as did the "texture" of the nuclear placement, based on two run-length statistics. Combination of the run-length features distinguished the three categories of lesions with statistical significance. The results of this study provided insights into the problems (such as the effect of field boundaries) faced in the design of an computer-aided grading system. They also showed the value of expert system-guided scene segmentation and of such histometric features as the field cellularity and the number of nuclei per gland for the discrimination between lesions of different grades of differentiation.     

Lactic acid bacteria: model systems for in vivo studies of sugar transport and metabolism in gram-positive organisms

Lactic acid bacteria provide a model system for the in vivo study of mechanisms pertaining to the regulation of sugar transport and metabolism by microorganisms. Recent studies with resting and growing cells of the homofermentative Streptococci and Lactobacilli have yielded evidence for hitherto unsuspected regulatory mechanisms in this group of industrial and medically important bacteria. These regulatory mechanisms mediate the exclusion and expulsion of sugars, the preferential transport of sugar from sugar mixtures, resistance to non-metabolizable sugar analogs and participate in the establishment of energy-dissipating futile cycles. Transport experiments conducted with novel sugar analogs, data from enzymatic analyses and 31P-NMR spectroscopy studies with wild type and mutant strains of Streptococci, have provided new insight into the fine- and coarse-controls responsible for the modulation of activity of the sugar transport: glycolysis cycle. The purpose of this review is to summarize our current knowledge of these regulatory mechanisms and to suggest avenues for future investigation. Although specifically addressed to the lactic acid bacteria, it seems likely that some of the mechanisms described will be found in other Gram-positive species.     

Role of cell-cycle in regulating neuroepithelial cell shape during bending of the chick neural plate

Summary Neuroepithelial cells transform from spindle-shaped to wedge-shaped within the median and paired dorsolateral hinge points of the bending neural plate, but the mechanisms underlying these localized changes are unclear. This study was designed to evaluate further the hypothesis that localized wedging of neuroepithelial cells during bending involves basal cellular expansion resulting from alteration of the cell-cycle. Neurulating chick embryos were treated with tritiated thymidine, and transverse sections through the midbrain were examined autoradiographically. Parameters of the cell-cycle as well as nuclear position and size were assessed in the median hinge point, which contains predominantly wedge-shaped cells, and in adjacent lateral areas of the neural plate, which contain predominantly spindle-shaped cells. Both the DNA-synthetic phase and non-DNA synthetic portion of the cell-cycle were significantly longer in the median hinge point than in lateral neuroepithelial areas, some nuclei in both regions were located basally during these phases, and virtually all basal nuclei in the median hinge point were large. Additionally, the mitotic phase was significantly shorter in the median hinge point than in lateral areas. We present a model to explain how alteration of the cell-cycle in the median hinge point could generate wedging of cells in this region.