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E C Cox

Publications and source records attributed to E C Cox.

At least 19 recordsLinked to original sources
PubMed1995

Green fluorescent protein production in the cellular slime molds Polysphondylium pallidum and Dictyostelium discoideum.

The green fluorescent protein-encoding gene from Aequorea victoria has been cloned into several different transforming vectors and expressed in the cellular slime molds, Polysphondylium pallidum and Dictyostelium discoideum. We find that the protein is stable and non-toxic in both species, can be easily visualized in living and fixed specimens, and can be used to purify rare cells by fluorescence-activated cell sorting (FACS).

Animals↗
PubMed1995

Development in one dimension: the rapid differentiation of Dictyostelium discoideum in glass capillaries.

When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps toward the formation of prestalk and prespore zones. Some of the events occur within a minute or two, whereas others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different-sized cell masses, and those proportions are the same as those found in normal slugs. When the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein expressed from a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells.

Alkaline Phosphatase↗
PubMed1995

Trapping of branched DNA in microfabricated structures.

We have observed electrostatic trapping of tribranched DNA molecules undergoing electrophoresis in a microfabricated pseudo-two-dimensional array of posts. Trapping occurs in a unique transport regimen in which the electrophoretic mobility is extremely sensitive to polymer topology. The arrest of branched polymers is explained by considering their center-of-mass motion; in certain conformations, owing to the constraints imposed by the obstacles a molecule cannot advance without the center of mass first moving a short distance backwards. The depth of the resulting local potential well can be much greater than the thermal energy so that escape of an immobilized molecule can be extremely slow. We summarize the expected behavior of the mobility as a function of field strength and topology and point out that the microfabricated arrays are highly suitable for detecting an extremely small number of branched molecules in a very large population of linear molecules.

Bacteriophage lambda↗
PubMed1992

Modeling and experiment in developmental biology.

Models in developmental biology continue to yield valuable insights, yet do not play a strong role as guides to experiment. This may be because of the largely unexplored complexity of most developing organisms, and the fact that the most powerful models work at a very abstract level. In Dictyostelium discoideum, however, a fully formed model incorporating detailed experimental results is now available.

Animals↗
PubMed1992

Establishment and maintenance of stable spatial patterns in lacZ fusion transformants of Polysphondylium pallidum.

Polysphondylium pallidum cells were transformed with a construct containing the Dictyostelium discoideum ecmA promoter fused to a lacZ reporter gene. Two stably transformed lines, one in which beta-galactosidase (beta-gal) is expressed in apical cells of the fruiting body (p63/2.1), and one in which it is expressed in basal cells (p63/D), have enabled us to infer how cells move during aggregation and culmination. Several types of cell movement proposed to occur during slime mold culmination, such as random cell mixing and global cell circulation, can be ruled out on the basis of our observations. Cells of the two transformant lines express beta-gal very early in development. In both cases, stained cells are randomly scattered in a starving population. By mid to late aggregation, characteristic spatial patterns emerge. Marked cells of p63/2.1 are found predominantly at tips of tight aggregates; those of p63/D accumulate at the periphery. These patterns are conserved throughout culmination, showing that marked cells maintain their relative positions within the multicellular mass following aggregation. Neither the apical nor the basal pattern appears to be regulated within the primary sorogen by de novo gene expression or by cell sorting as whorls are formed. However, marked cells within a whorl re-establish the original pattern in secondary sorogens. This must be achieved by cell migration, since beta-gal is not re-expressed.

Cell Movement↗
PubMed1990

Analysis of developmentally expressed antigens in Polysphondylium pallidum.

A series of monoclonal antibodies were previously raised against developing Polysphondylium pallidum cells. In this work, six of these antibodies have been used as probes to identify and characterize antigens regulated during development. Soluble and membrane fractions of P. pallidum cells at six stages of development or three stages of cyclic adenosine monophosphate (cAMP)-induced development were run in sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to Western blot analysis. Three of the monoclonals, anti-Tp200, anti-Tp423, and anti-Pg101, stain sorogen tips. Tp423 and Tp200 are membrane-associated antigens; both are stable to urea extraction, and Tp200 remains in the membrane after NaOH extraction. Tp423 is present in starved cells but is more prominent in sorogens and particularly in cAMP-developed cells. In contrast, Tp200 is first detected in early to mid-aggregation and is more abundant late in development. Pg101, which is expressed as a gradient with its highest concentration in tips, first appears in tight aggregates but is much more abundant in sorogens; unlike the Tp antigens, Pg101 is not greatly induced in cAMP-developed cells. All three of these antigens undergo changes in apparent molecular weight at the tight aggregate or sorogen stage: The gel mobilities of Tp200 and Pg101 increase, whereas that of Tp423 decreases. In addition to the tip-specific monoclonals, two monoclonals that stain all but the tips of sorogens have been used for analysis. One of these, anti-3D10Pnk stains most cells within secondary tips, whereas anti-3D10Dif does not. 3D10Dif is membrane associated; it is present very early in development, increasing two- to threefold through the sorogen stage and diminishing in late cAMP-developed cells. 3D10Pnk is a mostly soluble species first detected in late streaming. Anti-1c3, a sixth monoclonal, which stains nuclei uniformly throughout sorogens, is also developmentally expressed. 1c3 is mainly membrane associated and is expressed from late streaming through the sorogen stage.

Antigens, Fungal↗
PubMed1990

Biochemical characterization of a putative axonal guidance molecule of the chick visual system.

Temporal retinal axons growing in vitro on carpets of tectal membranes are deflected by cell membranes of posterior tectum. The activity responsible for this deflection can be abolished by antibodies raised against tectal membranes and the corresponding Fab fragments. Analysis of tectal membranes by two-dimensional gel electrophoresis and immunoblotting reveals a 33 kd glycoprotein that has a higher concentration in posterior than in anterior tectum. Its expression is developmentally regulated, and it is sensitive to phosphatidylinositol-specific phospholipase C. These are properties expected for a molecule responsible for the phenomena observed in experiments on in vitro guidance of retinal axons.

Animals↗
PubMed1990

Axonal guidance in the chick visual system: posterior tectal membranes induce collapse of growth cones from the temporal retina.

Membranes from posterior and anterior thirds of the chick optic tectum were added to explants from nasal and temporal retina. Posterior membranes, and to a lesser extent anterior membranes, cause temporal growth cones to collapse and their axonal processes to retract. Neither tectal source has an effect on nasal growth cones. We interpret these results to mean that there is a tectal activity, stronger in the posterior than the anterior region of the tectum, which helps guide growth cones during the development of the retinotectal map. We believe that in vivo this activity helps to steer temporal growth cones away from the posterior tectum. Nasal growth cones, which must map to the posterior tectum, are resistant to it. In vitro, when posterior membranes contact temporal growth cones over their surface, filopodia and lamellipodia withdraw rapidly. This leads to loss of contact between the growth cone and the substrate, followed by collapse.

Animals↗
PubMed1990

Electrophoretic karyotype for Dictyostelium discoideum.

This paper reports on the separation of the Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.

Blotting, Southern↗
PubMed1988

Geometry and spatial patterns in Polysphondylium pallidum.

The formation of secondary sori in whorls of Polysphondylium pallidum provides an attractive model system for the study of symmetry breaking during morphogenesis. Tip-specific antibodies that permit detection of very early stages in this patterning process are available. We have found that the patterns of tip-specific antigen expression vary considerably depending on the size, shape, and developmental stage of the whorl. All of these patterns, however, are well explained by patterning models that rely on short-range autocatalysis and long-range inhibition, as exemplified by reaction-diffusion theories. In the context of reaction-diffusion, we discuss the possible effects of initial conditions, boundary conditions, and nonlinearities on the selection of patterns in P. pallidum whorls.

Models, Theoretical↗
PubMed1988

Spatial patterns in the fruiting bodies of the cellular slime mold Polysphondylium pallidum.

During morphogenesis in the slime mold Polysphondylium pallidum cell masses are periodically pinched off from the base of the developing sorogen. These masses round up and differentiate into secondary sorogens, which become radially ordered arrays of secondary fruiting bodies called whorls. Here we describe the morphogenesis of P. pallidum and characterize the spacing of whorls along the central stalk of the fruiting body and the spacing of sorocarps within whorls. We find both are highly regular. We propose that the linear spacing of whorls can be accounted for satisfactorily by a model that views the periodic release of cell masses from the base of the developing sorogen as the consequence of an imbalance between forces that orient amoebae toward the tip of the culminating sorogen, and cohesive forces between randomly moving cells in the basal region of the sorogen, which act as a retarding force. The orderly arrangement of fruiting bodies within whorls can be explained most easily by models that employ short-range activation and lateral inhibition.

Cell Differentiation↗
PubMed1987

Genesis of a spatial pattern in the cellular slime mold Polysphondylium pallidum.

The branches in Polysphondylium pallidum whorls are arranged in a radial pattern. We have used a pattern-specific monoclonal antibody to study branch formation and characterize the origin of this pattern. A quantitative spatial analysis of antibody staining reveals that the branching pattern arises from a random distribution. This distribution passes through a series of intermediate stages to yield a radial prepattern. The origins and evolution of this prepattern are satisfactorily accounted for by models that produce spatial patterns by short-range autocatalytic and longer-range inhibitory forces.

Antibodies, Fungal↗
PubMed1986

DNA sequence and coding properties of mutD(dnaQ) a dominant Escherichia coli mutator gene.

The mutD(dnaQ) gene in Escherichia coli codes for the epsilon subunit of the DNA polymerase pol III holoenzyme. Previous work has shown that this gene lies adjacent to the gene coding for RNase H (rnh). The two products are translated from diverging promoters. Here we report on the 1.6 kb (1 kb = 10(3) bases or base-pairs) sequence of the region coding for both genes, and the transcripts encoded by them. mutD codes for two transcripts, one of whose origins lies within the rnh structural gene. Both transcripts overlap and are complementary to a region of the rnh transcript. Thus, they can potentially form double-stranded helices with rnh. Of the two possible double-stranded structures, the shorter does not interfere with a likely rnh ribosome binding site, while the longer one does. We suggest that this unique organization may regulate rnh translation rates.

Amino Acid Sequence↗
PubMed1986

Spatial patterning in Polysphondylium: monoclonal antibodies specific for whorl prepatterns.

In this report we describe three monoclonal antibodies which detect prepatterning events preceding the appearance of visible tips in Polysphondylium pallidum whorls. A spatial and temporal analysis of the antigens against which these antibodies are directed reveals that the radial distribution of arms within whorls has its origins in an initial global amplification of tip-specific antigens over the surface of very early whorl masses. This two-dimensional distribution becomes restricted with time to a single dimension, a smooth distribution of antigen in a band about the equator of the whorl mass. This equatorial distribution breaks up into patches which eventually become visible tips. These results reveal that a spatial pattern can arise from a smooth prepattern, and grow through a series of intermediates characteristic of the symmetry breaking model first described by A. Turing (1952).

Antibodies, Monoclonal↗
PubMed1983

Structure and coding properties of a dominant Escherichia coli mutator gene, mutD.

The region of the Escherichia coli chromosome coding for the mutD gene was cloned, mutD5 function resides on a 1.2-kilobase fragment coding for a 28-kilodalton (kDa) protein. A deletion end-point analysis shows that the presence of the 28-kDa protein is required for mutD5 function and suggests that the mutD functional region has sufficient capacity to code for a second polypeptide of approximately 20 kDa. Plasmids carrying the mutD5 and mut+ alleles both produce the 28-kDa species. The product of mutD5 is dominant when carried by single and multicopy plasmids. The product of mut+ is dominant only on multicopy plasmids. Thus, mutD5 exhibits negative complementation. We suggest that the 28-kDa protein participates in a multimeric structure, perhaps at the replication fork.

DNA, Bacterial↗
PubMed1982

Dominant mutators in Escherichia coli.

In this paper we report on the isolation and genetic analysis of a series of strong mutators mapping at five minutes on the E. coli chromosome. These mutations are dominant and show no evidence of interaction in merodiploids. Cultures grown in broth medium exhibit mutant frequencies five to six orders of magnitude higher than mut+ strains. Cultures propagated in minimal salts media mutate at rates one to three orders higher than wild-type. Three-factor crosses have been used to order these mutators relative to metD, proA, and a Tn10 insertion near five minutes.

Chromosome Mapping↗
PubMed1980

Interaction of an Escherichia coli mutator gene with a deoxyribonucleotide effector.

Strains of Escherichia coli carrying mutD5 display very high mutation rates (about 10(4)-fold above wild-type) when grown in rich medium, but relatively low mutation rates (about 10- to 50-fold above wild-type) in minimal medium. Thymidine, deoxycytidine, and deoxyuridine are all capable of stimulating mutation when added to minimal medium. Studies with mutants blocked in various steps of thymidine metabolism implicate a phosphorylated thymidine effector which mediates the mutagenic action of the added deoxyribonucleotides. In addition, an unidentified compound or compounds other than thymidine present in rich medium (L-broth) can also increase the mutation rate.

Culture Media↗