Brigitte Rosenwirth

Insertion mutations in the control region of the galactose operon of E. coli

SummaryGalactose negative mutations are described which reduce the maximum expression of all three gal genes about 100-fold. The residual enzyme synthesis is not or only slightly inducible.These pleiotropic mutations map in the control region of the gal operon. No recombination is observed between these mutations. All mutants revert spontaneously to a Gal+ phenotype. In some mutations wildtype-like as well as constitutive revertants are obtained. The frequency of reversion can be increased by nitrosoguanidine (NG) in all mutants. The revertants, induced by this mutagen, are of a constitutive type.

Early processes of echovirus 12-infection: elution, penetration, and uncoating under the influence of rhodanine.

Abstract The early events in the interaction of radioactively labeled, highly purified echovirus 12 with green monkey kidney cells (GMK line) are analyzed in the absence and presence of rhodanine, 2-thio-4-oxothiazolidine, a specific inhibitor of echovirus 12 uncoating. In the presence of rhodanine, elution of modified viral particles is impaired; furthermore, after release of the rhodanine block no elution takes place. In the absence of rhodanine, however, i.e., in normal infection, elution of noninfectious A particles is observed. Apparently, after incubation at 36° in the presence of rhodanine a large proportion of the virus has reached a location—possibly the cytoplasm—from where it cannot be sloughed into the medium. We conclude that in the presence of rhodanine penetration of intact virus takes place, and modification to noninfectious particles is not a necessary precondition for penetration. The infection of these penetrated particles, however, appears to be abortive, since upon removal of rhodanine only a small proportion of cell-associated intact virus is uncoated. In vitro modification of echovirus 12 leads to the formation of particles similar to the described in vivo intermediates of uncoating. Rhodanine is demonstrated to stabilize the virion against heat inactivation and thus inhibits creation of heat degradation products. A generalized hypothesis to explain the mode of action of rhodanine during the early processes of echovirus 12 infection is proposed.

Incomplete particles of adenovirus: II. Kinetics of formation and polypeptide composition of adenovirus type 2

Abstract When KB cells growing in suspension culture are infected with adenovirus type 2 (Ad 2) 5–15% of the virus particles formed are incomplete in that they contain only fragments of viral DNA. The kinetics of appearance of incomplete particles and complete virions have been determined by measuring the amount of each type of particles produced at various times after infection. The incomplete particles can be detected starting 13 hr after infection. The percentage of incomplete particles is independent of the multiplicity of infection used. A comparison of the polypeptide composition of the incomplete particles with that of the complete virions by electrophoresis on polyacrylamide slab gels demonstrates that both types of particles contain hexons, penton base and fibers in similar relative amounts. However, the incomplete particles lack the core proteins (polypeptides V and VII) and contain a number of polypeptides which are not apparent in the complete virions or, are present only in minor amounts.

Biochemistry and pathogenicity of echovirus 9 I. Characterization of the virus particles of strains Barty and Hill

Abstract Strain Barty, a mouse-pathogenic echovirus 9, which is known to cause epidemic disease in man, and strain Hill, the prototype of echovirus 9, were characterized and compared. Strain Barty virion particles sediment in a low-salt-sucrose gradient with 156 S, whereas particles of the Hill strain form fast-sedimenting aggregates. Plaque formation of the Hill strain is inhibited under an overlay containing agar; virus strain Barty, however, forms plaques. Isoelectric focusing revealed that Barty virion particles exist essentially in one conformation with a characteristic isoelectric point; virions of the Hill strain were found to behave like a mixture of particles with various isoelectric points. Thus, the two echovirus 9 strains, apparently, have a different capsid surface. These divergent capsid surface structures include not only the adsorption site(s)-strain Barty adsorbs less efficiently to cells in culture than strain Hill-but also the determinants inducing neutralizing antibodies: serologically, the two strains are clearly distinguishable. The capsid protein patterns of the two strains in SDS-PAGE show a characteristic difference: VPl of strain Barty is a double band, whereas strain Hill contains a single band in a position just between the two VP1 bands of strain Barty. In addition, fingerprinting to the capsid proteins revealed that the two echovirus 19 strains differ not only in VP1 but probably also in VP3. These determinants of capsid proteins may cause the observed divergent behaviors of the two virus strains, and-possibly-influence pathogenicity.

Isolation and characterization of an arildone-resistant poliovirus 2 mutant with an altered capsid protein VP1

Arildone (4-[6-(2-chloro-4-methoxyphenoxy)hexyl] -3,5-heptanedione is a selective inhibitor of poliovirus uncoating. We have isolated an arildone-resistant poliovirus 2 variant and characterized it. In single cycle growth curve experiments the resistant virus was not influenced by 1 microgram/ml of arildone, which completely inhibited the replication of the sensitive virus. In neutralization experiments the rates of inactivation vs time of the parent and the resistant virus proved indistinguishable. The adsorption behavior of the mutant closely resembled that of the wild-type. Uncoating as measured with neutral red-sensitized arildone-resistant mutant virus was unaffected by arildone, in contrast to the uncoating of the sensitive parent virus. It has been previously shown that arildone stabilizes the poliovirus capsid from alkaline degradation. The arildone-resistant mutant, however, was not markedly stabilized by the drug under these conditions. No difference in thermostability between mutant and parent virus could be detected. In isoelectric focusing the sensitive virus exhibited 2 peaks, but in the presence of arildone only one peak was apparent. In contrast, the arildone-resistant virus remained unaffected by the presence of arildone. Analysis of the capsid proteins of the sensitive parent and the resistant virus in SDS-polyacrylamide gels revealed no difference in the patterns; however, peptide mapping showed clear differences in VP1.

Isolation and characterization of temperature-sensitive mutants of echovirus 12.

Abstract Thermosensitive mutants of echovirus 12 were isolated after mutagenization with 5-fluorouracil. Twelve of them were characterized in terms of the physiological defects that appear under restrictive conditions. At the nonpermissive temperature only 2 of the 12 mutants are able to synthesize viral RNA and protein in near-to-wild type amounts; however, no viral or subviral particles could be detected in cells infected with these two mutants. The remaining 10 mutants were found to be impaired in viral RNA synthesis. Six of the twelve mutants produced virus particles which were more thermolabile in vitro than those of the wild type; it is concluded that they carry a mutation in the gene(s) coding for structural protein. All of them belong to the class of mutants which were defect in viral RNA synthesis. These results suggest an involvement of structural protein in viral RNA synthesis.

A new class of mutants with an elevated basal level of the galactose enzymes in E. coli.

SummaryA new type of mutation is described which affects the regulation of the gal-operon, and maps distant from the structural genes and the galR-gene. The mutants were found among the survivors of a heat induction of the thermosensitive prophage λcI857. Their basal enzyme level is 2–3 times higher than the wild-type level. They carry deletions covering the region from attλ to chlA. The deletion exerts its influence in position trans, and the effect can be complemented. The presence of the deletion does not affect the enzyme level of the lac-operon.This evidence suggests that in the mutant strains an unknown gene is deleted, the product of which has some influence on the repression of the gal-operon. We propose galB (B for basal level) as a preliminary name for this gene.