Construction and properties of a chimeric bacteriophage lysis gene

Abstract The genomes of DNA phage ΦX174 and of RNA phage MS2 each encode a single lysis protein, E protein and L protein, respectively. Based on the known DNA and protein sequences, and with the aid of structural predictions of the proteins, a chimeric gene was constructed. The resulting protein was composed of the N-terminal sequence of E protein and the C-terminal sequence of L protein. The truncated E and L polypeptides used in this construction were functionally inactive. However, the chimeric gene product had very high lysis-inducing activity. This construction is an example which could be extended to the engineering of other lysis proteins designed with specific biotechnological processes in mind.     

Biochemical characterization of phi X174-protein-E-mediated lysis of Escherichia coli

Energetic and permeability properties of Escherichia coli cells were determined prior to and during lysis caused by expression of the cloned gene E of bacteriophage phi X174. Before onset of cell lysis the transmembrane gradients for K+, Na+ or Mg2+/ions, the level of ATP and the membrane potential, were unaffected. All these parameters changed simultaneously at the time of lysis onset, as monitored by measurements of culture turbidity as well as by determining the various specifications over a period of 1 min. During cell lysis chromosomal DNA was fragmented whereas plasmid DNA was liberated in its intact supercoiled form. Cytoplasmic constituents were released almost entirely, as indicated by the activity of beta-galactosidase in the supernatant fraction of protein-E-lysed cells. Periplasmic enzymes were only found in limited amounts in the cell supernatant and most remained associated with the cell ghosts. Such ghosts exhibited no gross cell damage or morphological alterations when compared with intact E. coli by light microscopy. All parameters investigated indicated that protein-E-mediated lysis of E. coli is caused by the formation of a transmembrane tunnel structure through the envelope complex of the bacterium.     

Proline 21, a residue within the α-helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

ΦX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded α-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli . Oligomerization of protein P21G-StrpA was not disturbed.     

Leu-enkephalin-stimulated growth hormone and prolactin release in the rat: comparison with the effect of morphine

The effect of Leu⁵-enkephalin on growth hormone (GH) and prolactin (PRL) release was studied $\text{in vivo}$ in the infant rat and compared to that of morphine. In 10 day-old pups, intracerebroventricular injection of Leu⁵-enkephalin (50, 75 and 100 μg) resulted in a dose-related increase in plasma GH; morphine was active as GH releaser at the dose of 5 and 10 μg, but not at 2.5 μg. Pretreatment with naloxone (2 mg/kg ip) suppressed the GH-releasing effect of either Leu⁵-enkephalin (100 μg) or morphine (10 μg). Leu⁵-enkephalin (75 and 100 μg) induced a rise in plasma PRL which was neither dose-related nor antagonized by naloxone; morphine (5 and 10 μg) was active as PRL releaser and its effect was antagonized by naloxone. These results indicate that: 1) Leu⁵-enkephalin stimulates both GH and PRL release; 2) the release of GH by Leu⁵-enkephalin but likely not that of PRL involves specific opiate receptors; 3) morphine releases GH and PRL through specific opiate receptors.     

Ultrastructure of the lateral-line sense organs of the ratfish,Chimaera monstrosa

Summary The ultrastructure of the lateral-line neuromasts in the ratfish, Chimaera monstrosa is described. The neuromasts rest at the bottom of open grooves and consist of sensory, supporting, basal and mantle cells. Each sensory cell is equipped with sensory hairs consisting of a single kinocilium and several stereocilia. There are several types of sensory hair arrangement, and cells with a particular arrangement form patches within the neuromast. There are two types of afferent synapse. The most common afferent synapse has a presynaptic body and is typically associated with an extensive system of anastomosing tubules on the presynaptic side. When the tubules are absent, vesicles surround the presynaptic body. These synapses are often associated into synaptic fields, containing up to 35 synaptic sites. The second type of afferent synapse does not have a presynaptic body and is not associated with the tubular system. The afferent synapses of the second type do not form synaptic fields and are uncommon. The efferent synapses are either associated with a postsynaptic sac or more commonly with a strongly osmiophilic postsynaptic membrane. The accessory cells are similar to those in the acoustico-lateralis organs of other aquatic vertebrates. A possibility of movement of the presynaptic bodies and of involvement of the tubular system in the turnover of the transmitter is discussed. A comparison of the hair tuft types in the neuromasts of Ch. monstrosa with those in the labyrinth of the goldfish and of the frog is attempted.     

Analysis of a temperature sensitive mutation in gene cII of bacteriophage lambda

Summary The mutation cIIts612 was found to map outside the immunity region of phage imm21 hybrid. As expected of a cII mutation, cIIts612 is unable to stimulate either cI repressor or Int synthesis during the establishment of lysogeny. These results indicate that part of the cII gene of is homologous to that of imm21 phage.