Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu

Casadaban, Malcolm J.

doi:10.1016/0022-2836(76)90119-4

Cited by 1,824 publications

(589 citation statements)

References 24 publications

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“…To construct E. coli WJT-1 cells, E. coli TK2313 (F -thi rha lacZ nagA ⌬(kdpAE81) trk1 trkA405 endoA) was transduced with P1 lysate of a thr34::Tn10 strain, and a tetracycline-resistant colony was purified. This strain was then transduced to Thr ϩ , araD139 with a lysate of strain MC4100 (14). One araD139 derivative of TK2313 was purified and transduced to ilv500::Tn10 with a lysate of strain MRi134 (pertinent marker is ilv500::Tn10), and a tetracycline-resistent colony was purified.…”

Section: Methodsmentioning

confidence: 99%

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Yan

Huang

Shaw

et al. 1997

Journal of Biological Chemistry

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Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C 2 domain is catalytically active and the C 1 domain is not; the role of the C 1 domain is to promote the catalytic activity of the C 2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C 2 domains, but not among the C 1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30 -100-fold reduction in K cat of adenylyl cyclase. However, the same mutations do not affect the K m for ATP, the half-maximal concentration (EC 50 ) for the C 2 domain of type II adenylyl cyclase to associate with the C 1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC 50 for forskolin to maximally activate enzyme activity with or without G s␣ . This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C 1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in K cat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.The activity of mammalian adenylyl cyclase, the enzyme that converts ATP to cAMP, is the key step in modulating intracellular cAMP concentration in response to extracellular stimulation by hormones, neurotransmitters, and odorants. Nine isoforms of mammalian adenylyl cyclases have been cloned to date, and they belong to a rapid expanding cyclase family that includes class III adenylyl cyclases 1 and guanylyl cyclases (1).All nine isoforms have a similar structure, including two intensely hydrophobic domains (M 1 and M 2 ) and two 40-kDa cytoplasmic domains (C 1 and C 2 ). The two cytoplasmic domains contain sequences (C 1a and C 2a ) that are homologous to each other and to class III adenylyl cyclases and guanylyl cyclases. Each isoform of adenylyl cyclase not only has distinct patterns of tissue expression but also has unique responses to extracellular and intracellular stimuli (1-3). In common, each adenylyl cyclase can be activated by the G protein 2 ␣ subunit, designated as G s␣ , and each can be inhibited by certain adenosine analogues (P-site inhibitors). However, there are several subtype-specific regulators, including forskolin, G protein ␤␥ subunits, the ␣ subunit of G i , G o , and G z , Ca 2ϩ ion, Ca 2ϩ -calmodulin, Ca 2ϩ -calcineurin, cAMP-dependent protein kinase, and PKC (for review, see Refs. 1-3). Certain ones of these regulators (e.g. ␤␥) can be either stimulatory or inhibitory...

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Section: Methodsmentioning

confidence: 99%

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Yan

Huang

Shaw

et al. 1997

Journal of Biological Chemistry

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“…My colleague Malcolm Casadaban had developed a technique using Mu to generate hybrid fusion proteins in vivo (Casadaban, 1976), and my former PhD student, Spencer Benson, piqued my interest in the fusion process when he told me it required thick agar plates because the fusion colonies only appeared after long periods of incubation. After failing to persuade Spencer to study this phenomenon, I undertook the subject myself and quickly found evidence that selection on the appropriate growth medium triggers a frequent Mudependent fusion process (found in over 1 out of every10 5 cells) which was undetectable during normal growth (found in fewer than 1 in every 10 10 cells; Shapiro, 1984a).…”

Section: Personal History: Transposable Elements Adaptive Mutation Amentioning

confidence: 99%

Bacteria are small but not stupid: cognition, natural genetic engineering and socio-bacteriology

Shapiro

2007

Studies in History and Philosophy of Science Part C: Studies in

172

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40 years experience as a bacterial geneticist have taught me that bacteria possess many cognitive, computational and evolutionary capabilities unimaginable in the first six decades of the 20 th Century. Analysis of cellular processes such as metabolism, regulation of protein synthesis, and DNA repair established that bacteria continually monitor their external and internal environments and compute functional outputs based on information provided by their sensory apparatus. Studies of genetic recombination, lysogeny, antibiotic resistance and my own work on transposable elements revealed multiple widespread bacterial systems for mobilizing and engineering DNA molecules. Examination of colony development and organization led me to appreciate how extensive multicellular collaboration is among the majority of bacterial species. Contemporary research in many laboratories on cell-cell signaling, symbiosis and pathogenesis show that bacteria utilize sophisticated mechanisms for intercellular communication and even have the ability to commandeer the basic cell biology of "higher" plants and animals to meet their own needs. This remarkable series of observations requires us to revise basic ideas about biological information processing and recognize that even the smallest cells are sentient beings.

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“…Expression of the chromosomal lolA gene was induced with 1 mM IPTG. MC4100 [14] was used to prepare spheroplasts as described [4]. E. coli strains were grown at 37 ‡C on L-broth or M63 minimal medium supplemented with 0.2% maltose.…”

Section: Bacteria and Mediamentioning

confidence: 99%

Dominant negative mutant of a lipoprotein‐specific molecular chaperone, LolA, tightly associates with LolCDE

2002

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Periplasmic molecular chaperone LolA and the inner membrane ATP binding cassette transporter LolCDE are essential for ATP-dependent release of outer membrane-directed lipoproteins from the inner membrane of Escherichia coli. A LolA(F47E) mutant carrying a Phe to Glu mutation at position 47 was defective in the release of lipoproteins from spheroplasts and proteoliposomes reconstituted with LolCDE. When incubated with proteoliposomes containing LolCDE, LolA remained in the supernatant whereas LolA(F47E) bound to proteoliposomes. This tight association of LolA(F47E) with LolCDE caused a dominant negative phenotype in vivo, suggesting that the LolA^LolCDE interaction is critical for lipoprotein release. ß

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Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu

Cited by 1,824 publications

References 24 publications

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Bacteria are small but not stupid: cognition, natural genetic engineering and socio-bacteriology

Dominant negative mutant of a lipoprotein‐specific molecular chaperone, LolA, tightly associates with LolCDE

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