The Missing Discipline: Teaching K-12 Art History

Abstract

The Ecology of Imagination

Edith Cobb. The Ecology of Imagination in Childhood. Spring Publications, 1993. 139 pages. $dollar;15 paperback.     

EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12.

The regulatory proteins OmpR and EnvZ are both required to activate expression of the genes for the major outer membrane porin proteins, OmpF and OmpC, of Escherichia coli K-12. Here we show that OmpR, under certain conditions, could activate porin expression in the complete absence of EnvZ. In addition, the pleiotropic phenotypes conferred by a particular envZ mutation (envZ473) required the presence of functional OmpR protein. These results lead us to conclude that EnvZ and OmpR act in sequential fashion to activate porin gene expression; i.e., EnvZ modifies or in some way directs OmpR, which in turn acts at the appropriate porin gene promoter.     

12th international TNF conference: The good,the bad and the scientists

Members of the TNF superfamily control numerous aspects of immune defense as well as various processes of homeostasis and embryonic development. Recent advances in our knowledge of both the beneficial and the deleterious activities of these cytokines were thoroughly discussed at this conference. Participants presented new information about signaling mechanisms that these cytokines activate, with special attention to cell-death regulation, ubiquitination of signaling-proteins as a means of regulating their function, and complex systems of gene and signaling regulation. Sessions were devoted specifically to aberrations in functions of the TNF-family that contribute to the pathology of infectious, autoimmune and neurodegenerative diseases and to cancer, and to the application of our knowledge to therapy.     

The cloning of the Escherichia coli K-12 deoxyribonucleoside operon

A 6.1-kb EcoRI DNA fragment containing the four structural genes (deoC, deoA, deoB, deoD) of the deoxyribonucleoside operon has been cloned into the plasmid pMFS53. By use of a unique, asymmetrically positioned HindIII site on the 6.1 kb insert, plasmids containing the deoC,deoA genes (pMFS50) or the deoB,deoD genes (pMFS55) have been constructed. Enzyme assays performed on extracts prepared from clones harboring pMFS53, pMFS50 or pMFS55 revealed that each clone possessed amplified deo enzyme levels and that the spectrum of enzyme amplification corresponded to the genetic composition of the plasmids carried by each clone. A plasmid (pMFS50l) having functional deoA, deoB and deoD genes but devoid of the deo regulatory region and a portion of the deoC structural gene has been isolated following treatment of BamHI cleaved pMFS53 and BAL31 nuclease. Comparison of the deo enzyme levels for clones harboring pMFS53 and pMFS501 suggest that plasmid pMFS53 possesses a functional deo regulatory region in addition to the four structural genes of the operon.     

Improved bacterial baby machine: application to Escherichia coli K-12.

Exponentially growing derivatives of Escherichia coli K-12 were immobilized onto the surfaces of nitrocellulose membrane filters which had been coated with poly-D-lysine. The cells attached firmly to the surfaces, and when flushed with culture medium, the immobilized cells continued to divide and newborn cells were released into the effluent. Cell cycle parameters were examined with the technique, and it was found that K-12 derivatives possessed differing values for interdivision times, C, D, and average cell sizes when grown in the same culture media. It was also found that the cells released from immobilized populations of one culture consisted of two predominant size classes: newborn cells of unit size with single nucleoids and newborn cells of double this unit size. The results demonstrated that K-12 derivatives can be used in the baby machine culture technique to examine all aspects of the cell cycle of this organism. Furthermore, the yield of newborn cells was about fivefold greater than that obtained previously with cultures of strain B/r immobilized onto uncoated membranes.     

Heat damage to the chromosome of Escherichia coli K-12.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after in vivo heat treatment. Heat treatment of cultures at 50 degree C for 15, 30, and 60 min resulted in in vivo association of the nucleoids with cellular protein. Structural changes, determined by the increase in speed dependence of the nucleoids from heated cells, also occurred. These changes were most likely due to the unfolding of the typical compact nucleoid structure. The nucleoids from heated cells also had notably higher sedimentation coefficients (3,000 to 4,500S) than nucleoids from control cells (1,800S). These nucleoids did not contain greater than normal amounts of membrane phospholipids or ribonucleic acid. We propose that the protein associated with the nucleoids from heated cells causes the observed sedimentation coefficient increases.     

The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns. All guaA strains, including three frameshift mutants, produced derepressed levels of IMP dehydrogenase when cultured with a growth-limiting concentration of guanine. The guaB strains were of two types: (i) those producing derepressed levels of XMP aminase, and (ii) those producing basal levels of XMP aminase when grown under conditions of guanine starvation. In the guaB strains of the second type, the expression of the adjacent guaA gene is reduced. It is proposed that this pleiotropic effect of some guaB mutations is a result of polarity. The orientation of polarity suggests the gene order "operator"-guaB-guaA. Gel diffusion studies with IMP dehydrogenase antiserum showed that strains carrying polar guaB mutations do not produce cross-reacting material (CRM). The remaining guaB mutants were either CRM(+) or CRM(-). Mapping the mutations by three-factor crosses showed that polar and nonpolar guaB sites are clustered in a small genetic region cotransducible with guaA. The relative positions of the guaB mutational sites established that the polar mutations lie within the structural gene for IMP dehydrogenase.     

The WASP-WAVE protein network: connecting the membrane to the cytoskeleton

Wiskott-Aldrich syndrome protein (WASP) and WASP-family verprolin-homologous protein (WAVE) family proteins are scaffolds that link upstream signals to the activation of the ARP2/3 complex, leading to a burst of actin polymerization. ARP2/3-complex-mediated actin polymerization is crucial for the reorganization of the actin cytoskeleton at the cell cortex for processes such as cell movement, vesicular trafficking and pathogen infection. Large families of membrane-binding proteins were recently found to interact with WASP and WAVE family proteins, therefore providing a new layer of membrane-dependent regulation of actin polymerization.     

Cell polarity: connecting to the cortex

A GTPase module controls growth-site selection in budding yeast cells. The GDP--GTP exchange factor of this module, Bud5, has now been localized to sites of cell division and shown to interact with a transmembrane protein that marks these sites.     

The DnaA protein determines the initiation mass of Escherichia coli K-12

DNA replication was studied in a dnaA(Ts) strain containing a plasmid with the dnaA+ gene under plac control. At 42 degrees C, initiation of DNA replication was totally dependent upon the gratuitous inducer isopropyl beta-D-thiogalactopyranoside (IPTG). Flow cytometric measurements showed that at 13% induction of the lac promoter the growth rate, cell size, DNA content, and timing of initiation of DNA replication were indistinguishable from those observed in a wild-type control cell. Higher levels of induction resulted in initiations earlier in the cell cycle and a corresponding increase in the time from initiation to termination. We conclude that the concentration of DnaA protein determines the time of initiation and thereby the initiation mass. With an induction level equal to or above 13%, the synchrony of multiple initiations within one cell was close to that found in a wild-type control cell, showing that a cyclic variation in DnaA content is not necessary for a high degree of synchrony.     

Outsider to insider: resetting the natural host niche of commensal E. coli K-12

The status of E. coli K-12 as an exclusively non-invasive, non-pathogenic bacterium has almost been incontrovertible. Our recent finding that a mutation in one of its main architectural protein, HU, converts E. coli K-12 to an actively invasive form suggests that gaining host cell entry might be an expedient survival tactic for traditional commensals during certain altered host conditions. The mutant E. coli (SK3842) exhibits properties usually associated with pathogenic bacteria: host cell invasion, phagosomal disruption and intracellular replication. However, unlike the situation with some pathogens, internalized SK3842 imparts anti-apoptotic and cyto-protective effects rather than lethality on the host cell, both in vitro and in vivo. Here, we show that SK3842 also provides colonization resistance against other invasive pathogens--a trait not shared by the parental commensal strain. Thus, the altered lifestyle of SK3842 encompasses characteristics both from traditional pathogens as well as beneficial probiotic strains.     

Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system

The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1-nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system.     

Delivery of protein to the cytosol of macrophages using Escherichia coli K-12

Listeriolysin O (LLO) is an essential determinant of pathogenicity whose natural biological role is to mediate lysis of Listeria monocytogenes containing phagosomes. In this study, we report that Escherichia coli expressing cytoplasmic recombinant LLO can efficiently deliver co-expressed proteins to the cytosol of macrophages. We propose a model in which subsequent or concomitant to phagocytosis the E. coli are killed and degraded within phagosomes causing the release of LLO and target proteins from the bacteria. LLO acts by forming large pores in the phagosomal membrane, thus releasing the target protein into the cytosol. Delivery was shown to be rapid, within minutes after phagocytosis. Using this method, a large enzymatically active protein was delivered to the cytosol. Furthermore, we demonstrated that the E. coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the major histocompatibility (MHC) class I pathway for antigen processing and presentation, greater than 4 logs compared with E. coli expressing OVA alone. Moreover, the time required for processing and presentation of an OVA-derived peptide was similar to that previously reported when purified OVA was introduced directly into the cytosol by other methods. Using this system, potentially large amounts of any protein that can be expressed in E. coli can be delivered to the cytosol without protein purification. The potential use of this system for the delivery of antigenic protein in vivo and the delivery of DNA are discussed.     

Linkage Map of Escherichia coli K-12, Edition 10: The Physical Map

A physical map, EcoMap10, of the now completely sequenced Escherichia coli chromosome is presented. Calculated genomic positions for the eight restriction enzymes BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, and PvuII are depicted. Both sequenced and unsequenced Kohara/Isono miniset clones are aligned to this calculated restriction map. DNA sequence searches identify the precise locations of insertion sequence elements and repetitive extragenic palindrome clusters. EcoGene10, a revised set of genes and functionally uncharacterized open reading frames (ORFs), is also depicted on EcoMap10. The complete set of unnamed ORFs in EcoGene10 are assigned provisional names beginning with the letter “y” by using a systematic nomenclature.     

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12.

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.     

Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map

This map is an update of the edition 9 map by Berlyn et al. (M. K. B. Berlyn, K. B. Low, and K. E. Rudd, p. 1715–1902, in F. C. Neidhardt et al., ed., Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed., vol. 2, 1996). It uses coordinates established by the completed sequence, expressed as 100 minutes for the entire circular map, and adds new genes discovered and established since 1996 and eliminates those shown to correspond to other known genes. The latter are included as synonyms. An alphabetical list of genes showing map location, synonyms, the protein or RNA product of the gene, phenotypes of mutants, and reference citations is provided. In addition to genes known to correspond to gene sequences, other genes, often older, that are described by phenotype and older mapping techniques and that have not been correlated with sequences are included.     

The primary structure of the DeoR repressor from Escherichia coli K-12.

The nucleotide sequence of the deoR gene of E. coli, which codes for the DeoR repressor, has been determined. This gene codes for a polypeptide that is 252 amino acids residues in length. Computer-assisted analysis of the nucleotide sequence strongly suggests that the DNA binding domain of the DeoR repressor is located in the N-terminal part of the protein. After the coding region there is a dyad symmetry similar to a palindromic unit present outside many structural genes on the E. coli chromosome.