1,721,029 research outputs found
Development of multi-layer crystal detector and related front end electronics
No full textA crystal (diamond) particle detector has been developed and tested, whose constitute elements are a multi-layer polycrystalline diamond and a pick-up system capable of collecting in parallel the charge produced in the layers. The charge is read with a charge-to-voltage amplifier (5–6 mV/fC) realized with bipolar junction transistors in order to minimize the effect of the detector capacitance. The tests performed with cosmic rays and at the beam test facility of Frascati with 500 MeV electrons in single electron mode operation have shown that a detector with 4–5 layers of 250 μm thickness each and 9 mm2 active area exhibits an upper limit of 150 ps time resolution for minimum ionizing particles at an operating voltage of about 350 V
Two-hybrid assay: construction of an Escherichia coli system to quantify homodimerization ability in vivo
No full textA hybrid system which takes advantage of the properties of the lambda repressor allows detection of protein-protein interactions. Fusion of the cI N-terminal domain to a heterologous protein will result in a functional lambda repressor, able to strongly bind to its operator and conferring immunity to lambda infection only when the heterologous protein dimerizes efficiently. In this paper, construction of a recombinant plasmid which allows detection of the activity of the lambda chimeric repressor formed by the N-terminal part of cI fused with a heterologous protein is reported. This construct is interesting due to its potential to be integrated in any target gene of the bacterial host, thus permitting this hybrid assay to be performed, not only in Escherichia coli strains, but in every bacterial genus where the reporter gene can be expressed. In addition, because of its modular construction, this plasmid can be easily modified to be exploitable in many experimental situations, such as in the detection of promoter region activity
Mutations arise independently of transcription in non-dividing bacteria
No full textIt has been proposed that transcription introduces a bias into the random process of mutation. Although this hypothesis is supported by experimental data for mutations arising during active bacterial growth, the role of transcription in mutagenesis in non-dividing bacteria is entirely hypothetical. In the present study, we tested the hypothesis of a possible role of transcription in a non-dividing E. coli K12 strain. In this strain (BD010), a mutated trpB allele (trpB9578), placed under stringent transcriptional control, was tested for the appearance of prototrophic revertants on synthetic medium lacking tryptophan. The number of phenotypic revertants which appeared in the absence of trp transcription was compared to that observed when the mutated gene was continuously transcribed. Our results showed that transcription of trpB is not mutagenic under conditions of tryptophan starvation, and that the frequency of TrpB+ reversion is solely a function of the duration of starvation
A two-hybrid system based on chimeric operator recognition for studying protein homo/heterodimerization in Escherichia coli
No full textThe development of a convenient and promising alternative to the various two-hybrid methods that are used to study protein-protein interactions is described. In this system, a lambdoid chimeric operator is recognized by a hybrid repressor formed by two chimeric monomers whose C-terminal domains are composed of heterologous proteins (or protein domains). Only if these proteins efficiently dimerize in vivo is a functional repressor formed able to bind the chimeric operator and shut off the synthesis of a downstream reporter gene. This new approach was tested with several interacting proteins ranging in size from less than 100 to more than 800 amino acids and, to date, no size or topology limit has been detected
A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts
No full textEscherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac+ by prophage precise excision with a relatively high frequency (about 1 x 10(-6)). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced beta-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu
FtsZ dimerization in vivo
No full textA hybrid assay, based on the properties of the lambda repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N-terminal end of the lambda cI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional lambda repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N-terminal of about 150 amino acids; the C-terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ
Two-hybrid assay in Escherichia coli K12
No full textMany manifestations of living organisms are the result of protein activities that need the formation of the protein multimers themselves or of macromolecular complexes with other proteins. Numerous in vivo and in vitro approaches have been developed to facilitate the study of protein interactions [1–3]
Synchronous division induced in Escherichia coli K12 by gemts mutants of phage Mu.
No full textInfection with the bacteriophage mutant Mu c+ gemts2 at 42 degrees C induces synchrony in cell division in cultures of Escherichia coli K12. This synchrony may last for several cycles and is not only due to selection since synchronization is observed even when bacterial survival to the infection is over 80% as in lysogens for Mu c+ gemts2. The mechanism by which synchrony is induced is not known, but since the product of Mu gene gem (previously called lig) has been shown to interact with the enzymatic system in the bacteria controlling the degree of DNA supercoiling, the phenomenon could be a consequence of this interaction
The bacteriophage Mu gem gene: a positive regulator of the C operon required for normal levels of late transcription
No full textThe gem product of bacteriophage Mu modulates synthesis of various host proteins and alters the host chromosome topology. To elucidate the role of the gem gene in Mu development, we analyzed the behavior of several mutants in this gene. The results, obtained with two Mu gem- phages, show that (1) phage growth is significantly delayed and inhibited, (2) early transcription is normal but late transcription is delayed and reduced, (3) DNA replication appears normal, and (4) the Mu C gene, whose product positively regulates Mu late genes, is one of the gem target sites. Transcription of a C promoter-lacZ fusion, carried by the pPH91 plasmid, is stimulated both after infection with Mu gem+ or Mu gem3 and is strains lysogenic for the same phages in the presence of viral immunity. These data suggest that the primary role of the gem product is modulation of gene expression. This control could be carried out by direct interaction with transcription factors or by changing DNA supercoiling
- …
