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Biomedical subjects

J A Shapiro

Publications and source records attributed to J A Shapiro.

At least 55 records · Page 3Linked to original sources
PubMed1992

Pattern and control in bacterial colony development.

As we learn more about bacterial life in the laboratory and in nature, we increasingly appreciate that they are highly sensitive and sophisticated organisms. One of the principal new insights has been the appreciation that bacteria are interactive and form organized, differentiated multicellular communities. Colonies produced on laboratory media by the standard research bacterium, Escherichia coli, are excellent examples. The organization of these colonies can be visualized in the microscope, by macrophotography, and by the use of special dyes and genetic engineering techniques to reveal patterns of differential gene expression. Observation of the dynamics of colony growth, and the response of colonies to experimental disruptions of normal development, indicate that control systems work to produce the regular patterns observed. The effects of obstacles and of other colonies on gene expression patterns indicate that non-linear responses to chemical gradients in the substrate play an important coordinating role in colony development.

Bacteria↗
PubMed1990

Action of a transposable element in coding sequence fusions.

The original Casadaban technique for isolating fused cistrons encoding hybrid beta-galactosidase proteins used a Mucts62 prophage to align the upstream coding sequence and lacZ prior to selection. Kinetic analysis of araB-lacZ fusion colony emergence indicated that the required DNA rearrangements were regulated and responsive to conditions on selection plates. This has been cited as an example of "directed mutation." Here we show genetically that the MuA and integration host factor (IHF) transposition functions are involved in the formation of hybrid araB-lacZ cistrons and propose a molecular model for how fusions can form from the initial strand-transfer complex. These results confirm earlier indications of direct Mu involvement in the fusion process. The proposed model explains how rearranged Mu sequences come to be found as interdomain linkers in certain hybrid cistrons and indicates that the fusion process involves a spatially and temporally coordinated sequence of biochemical reactions.

Base Sequence↗
PubMed1989

General practitioners' attitudes towards AIDS and their perceived information needs.

A short questionnaire on general practitioners' self perceived and actual knowledge of AIDS and their attitudes to the illness was sent to 1824 general practitioners throughout the United Kingdom. The rate of response was 70%. Women doctors, those who trained overseas, and those who were married tended to have less positive attitudes towards patients with HIV and AIDS, whereas younger doctors, trainers, and members of the Royal College of General Practitioners were more understanding, better informed, and had more positive attitudes. Doctors with the least knowledge about HIV and AIDS and the most negative attitudes towards the illness would benefit from further education, which would be most effectively delivered through the professional journals, the Department of Health and the charitable AIDS organisations.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Rapid identification of microorganisms by nucleic acid hybridization after labeling the test sample.

A novel method for rapidly identifying microorganisms has been developed. This method employs a monoadduct-forming furocoumarin derivative, which can photochemically label nucleic acids. The labeled nucleic acid can, in turn, be hybridized simultaneously to a panel of immobilized probe DNAs arrayed as dots on a solid support such as nitrocellulose. This procedure offers several advantages over more conventional hybridization techniques in that sample nucleic acids can be photolabeled without substantial sample preparation and that identification can be achieved by a single, rapid hybridization reaction.

Bacterial Typing Techniques↗
PubMed1989

Escherichia coli K-12 cell-cell interactions seen by time-lapse video.

The high degree of organization in mature bacterial colonies suggests specific interactions between the cells during colony development. We have used time-lapse video microscopy to find evidence for cell-cell interactions. In its initial stages, Escherichia coli K-12 colony morphogenesis displayed control of the geometry of cell growth and involved intimate side-by-side associations. When microcolonies developed from isolated single bacteria, a directed process of elongation and division resulted in the appearance of a symmetrical four-cell array. When growth began with separate but nearby bacteria, the daughters of different cells elongated towards each other and also lined up side by side. Interactions between microcolonies containing several hundred or more bacteria were visible several hours later. Control of cell morphogenesis at later stages of microcolony development was strain specific. These results show that E. coli K-12 cells respond to each other and adjust their cellular morphogenesis to form multicellular groups as they proliferate on agar.

Cell Division↗
PubMed1989

Differential activity of a transposable element in Escherichia coli colonies.

In Escherichia coli colonies, patterns of differential gene expression can be visualized by the use of Mu d(lac) fusion elements. Here we report that patterned beta-galactosidase expression in colonies of strain MS1534 resulted from a novel mechanism, spatially localized replication of the Mu dII1681 element causing lacZ transposition to active expression sites. Mu dII1681 replication did not occur constitutively with a fixed probability but was dependent on the growth history of the bacterial population. The bacteria in which Mu dII1681 replication and lacZ transposition had occurred could no longer form colonies. These results lead to several interesting conclusions about cellular differentiation during colony development and the influence of bacterial growth history on gene expression and genetic change.

Blotting, Southern↗
PubMed1987

Expression of a synthetic gene encoding human insulin-like growth factor I in cultured mouse fibroblasts.

A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium from transfected cells inhibits binding of 125I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.

Amino Acid Sequence↗
PubMed1987

Organization of developing Escherichia coli colonies viewed by scanning electron microscopy.

Colony growth was initiated by inoculating minimal glucose agar with 1-microliter. spots of a plasmid-free Escherichia coli culture and incubating at 32 degrees C. Inoculations took place over a 3-day period, at the end of which the plates were fixed and dried for scanning electron microscopy. In this way, it was possible to examine the surfaces of colonies ranging in age from 0 to 68 h. Macroscopically, the colonies were organized into different concentric zones, and several morphological features could be seen to develop over this period. These included a shallow depression ring marking the site of inoculation, a deeper indentation ring whose position moved outward as the colony grew, an expanding plateau region between the two rings, a mound outside the indentation ring, and a flat brim extending onto the substrate which was either present or absent at different times. Microscopically, a variety of cell morphologies and cell arrangements were detected. Upon inoculation, the bacteria accumulated at the periphery of the inoculation spot but showed no other kind of order. For the first 7.5 h, all bacteria were rod shaped; at the end of this initial phase, a high degree of alignment was seen in the cells at the colony edge. By 24.5 h, both shorter more ovoid cells and longer filaments had begun to appear, and large multicellular arrays had formed. At later stages of colony development, morphologically distinguishable zones involving cells of different shapes and sizes had formed, and these zones often marked the boundaries of macroscopic features. The edges were particularly interesting and at 68 h displayed very sharp saw-toothed boundaries between concentrically organized groups of bacteria. There were some transient irregularities in the concentric organizations of growing colonies, and one colony had entered upon a distinct developmental pathway.

Escherichia coli↗
PubMed1985

Scanning electron microscope study of Pseudomonas putida colonies.

Pseudomonas putida colonies were examined by scanning electron microscope. A variety of cell morphologies, multicellular arrangements, and extracellular materials were observed in the fixed material. Different regions of a single colony showed characteristic organizations of these architectural elements. In some cases, the detailed microstructure of the fixed colony surfaces observed by scanning electron microscopy could be correlated with macroscopic patterns visualized by histochemical staining and surface relief photography of live colonies. Extracellular materials were seen to extend onto the agar surface beyond the boundaries of the cell mass, and the final structures of these materials, after fixation and desiccation, were colony specific. The significance of these features of colony microstructure for formulating hypotheses about the control of colony morphogenesis is discussed.

Microscopy, Electron, Scanning↗
PubMed1984

Physical structure, genetic content and expression of the alkBAC operon.

We cloned sequences of the alk (alkane utilization) operon of Pseudomonas and characterized them physically and genetically. These sequences were used to construct a DNA restriction map of the alkBAC region. We physically mapped alk::Tn7 insertions and delta alkBA deletions, and we were able to show complementation or marker rescue of alk point mutations by cloned DNA sequences. Our results confirmed the existence of an operon containing structural loci encoding activities for membrane alkane hydroxylase component (alkB), soluble alkane hydroxylase component (alkA) and membrane alcohol dehydrogenase (alkC). Physical mapping of alkC::Tn7 insertions and complementation of alkC point mutations by cloned sequences from the alkBA region showed that we were previously mistaken in inferring the existence of a separate unlinked alkC cluster. Studies with an alkB-lacZ transcription fusion construct established that the operon is transcribed in the order alkBAC and is under positive regulation by alkR regulatory functions.

Alkanes↗
PubMed1984

Reversal by DNA amplifications of an unusual mutation blocking alkane and alcohol utilization in Pseudomonas putida.

We analyzed the reversion of strains carrying alk208, a mutation in the alkBAC (alkane utilization) region of the Pseudomonas CAM-OCT plasmid. Reversion of alk208 was stimulated 25 to 75-fold by small doses of UV-irradiation. All alkane hydroxylase-positive (AlkB+) revertants proved to be aliphatic alcohol dehydrogenase-positive (AlkC+) as well, whereas AlkC+ revertants could be either AlkB+ or AlkB-. Most of the AlkB- AlkC+ partial revertants produced AlkC- segregants at measurable frequencies. UV-irradiation substantially increased the rate of AlkC- segregation. Most segregants reverted to AlkB+ or AlkC+ at frequencies similar to the original alk208 strain. Dot blot hybridization analyses using cloned probes from various regions of CAM-OCT revealed that the partial revertants contained specific amplications of alk DNA. The endpoints of these amplifications mapped in at least two regions. AlkC- segregants had lost the DNA amplifications.

Alcohols↗
PubMed1984

Observations on the formation of clones containing araB-lacZ cistron fusions.

Casadaban (1976) developed a technique for isolating E. coli clones containing fusions of the amino terminal-encoding portion of any cistron with the carboxy terminal-encoding portion of lacZ. The technique utilizes prophage Mu homology to bring the two cistrons into proximity. I have followed the appearance over time of colonies containing araB-lacZ fusions from a strain where the beginning of the araB cistron is connected to lacZ by an intact Mucts62 prophage. Cultures of the starting strain grown on a variety of media have fewer than 2 in 10(10) cells capable of forming colonies within three days after plating on selective arabinose-lactose medium. At 32 degrees C, there is a delay of between 4 and 19 days before the first colony appears. The kinetics of colony appearance over the next two to four weeks then shows a rapid increase in the number of new colonies emerging per day followed by a decline. The pattern of colonial emergence and the final numbers of fusion colonies obtained are not grossly affected by reducing the number of cells plated over five orders of magnitude. Fusion colonies sometimes show a clustered pattern when they first emerge. Inoculation of pre-existing fusion clones at specific locations on the arabinose-lactose selection plates seeded with the starting strain leads to the formation of inhibitory zones where no fusion colonies appear. Selection plates contain many microcolonies and papillae which do not proliferate into scoreable colonies but nonetheless contain cells capable of growth when replated on the same selective medium. Up to 39% of all plated cells are capable of producing fusion clones. The kinetics of fusion colony appearance can be altered by environmental and genetic manipulations. Partial derepression of the Mucts prophage at 37 degrees accelerates the appearance of colonies but also reduces the final yield. Addition of limiting concentrations of glucose to the selective medium also accelerates the appearance of colonies in a specific fashion: enrichments below the level required for maximum acceleration produce a biphasic kinetics with two waves of fusion clone emergence separated by an eight-day interval. Infection with Muc+pAp phage produces dilysogens that have almost completely lost the ability to produce fusions. Infection with MuctsAampAp phage produces strains that are reduced in phage production and have delayed kinetics of fusion clone emergence. The implications of these observations for theories of hereditary change in bacteria are discussed.

Bacteriophage mu↗
PubMed1984

Control of Tn7 transposition.

Our isolate of Tn7 (named Tn7S ) contains an IS1 insertion, and this IS1 can be converted into Tn9. In vitro and in vivo deletions of Tn7S and Tn7S ::Tn9 define regions of the transposon required for antibiotic resistance and transposition. Complementation of deletion mutants by cloned Tn7 fragments indicates the existence of two regions, denoted tnp7A and tnp7B , required for all transposition events. Another region, denoted tnp7C , is required for transposition from the chromosome to RP1 but not for transposition from a small IncP-1 replicon to the chromosome. The presence of Tn7S terminal sequences in an RP1 replicon reduces the transposition of a second Tn7S derivative from the chromosome by about one order of magnitude. The measured frequency of Tn7S transpositions from a small IncP-1 replicon to the chromosome depends on the particular incompatibility system used to eliminate that replicon. Genetic and physical data indicate that high frequencies of Tn7S transposition to the chromosome (greater than or equal to 40%) are triggered by the IncP-1 incompatibility reaction, thus suggesting the existence of a Tn7 mechanism for sensing the state of the carrier replicon.

Chromosomes, Bacterial↗
PubMed1984

The use of Mudlac transposons as tools for vital staining to visualize clonal and non-clonal patterns of organization in bacterial growth on agar surfaces.

When a histochemical stain for beta-galactosidase activity is applied to growth of Gram-negative bacteria on agar medium, the pigmentation is non-uniform and capable of revealing internal colony organization into different cell types. Use of an Escherichia coli strain with a thermosensitive lac repressor indicates that colonies expand by addition of new cells at the periphery and that older cells which have synthesized beta-galactosidase early in development remain in the centre. Mixed inocula of different strains show clonal exclusiveness as they proliferate outwards. Mudlac transposons can create genetic fusions that place beta-galactosidase expression under a variety of regulatory systems. Stained surface cultures of E. coli and Pseudomonas putida strains carrying Mudlac insertions in plasmids reveal a variety of flower-like staining patterns. These patterns display both clonal (i.e. sectorial) and non-clonal (circular and radial) features which are heritable within a given strain. The non-clonal aspects of the patterns reflect phenotypic differentiation without genetic change. These observations indicate that bacterial growth on agar surfaces is a highly regulated process similar, in many respects, to the development of specific multicellular tissues and organisms.

Agar↗