Isolation and characterization of mutants of bacteriophage T4 resistant to folate analogs

The dihydrofolate (FH₂) reductase specified by the T4 frd gene (previously called the wh gene) is apparently nonessential for phage growth on Escherichia coli because the bacterial FH₂ reductase can partially substitute for the phage enzyme. To study the effect in vivo of folate analogs which specifically inhibit the T4 FH₂ reductase, the enzyme must be made essential for phage production. Partial inhibition of the E. coli FH₂ reductase with the folate analog trimethoprim strongly inhibits phage production by a T4 frd mutant and slightly inhibits wild-type phage production. Adding an additional T4-specific folate analog, a chlorophenyl triazine, strongly inhibits wild-type phage production without preventing the uninfected E. coli cells from multiplying.We have isolated spontaneous mutants of T4 which are capable of producing progeny in the presence of chlorophenyl triazine and another folate analog pyrimethamine. These mutants are designated folate analog resistant (far) and have been separated into two general classes. Class I mutants induce T4-specific FH₂ reductases which are less sensitive to the action of the folate analogs than the normal FH₂ reductase. Class II mutants induce an unaltered FH₂ reductase but contain mutations in a variety of T4 genes. Some class II mutants induce more phage FH₂ reductase activity than wild-type T4 and appear to be regulatory mutants.     

Characterization of host-range and temperature-sensitive mutants of adenovirus type 5 with particular regard to transformation of a hamster embryo cell line (Nil)

Seventeen conditional lethal mutants (7 host-range and 10 temperature-sensitive) of adenovirus type 5 (Ad5) were classified by complementation test and characterized physiologically in viral-DNA synthesis, induction of cell DNA synthesis (in hamster kidney cells), capsid polypeptides production, and transformation of Nil cells (a hamster embryo cell line) under the restrictive conditions.Seven host-range (hr) mutants were divided into six groups by complementation test and into three classes by phenotypic characterization. Mutants assigned to class III (complementation groups D, E, F) were positive in viral-DNA synthesis and capsid polypeptides (hexon, penton base, fiber) production, and showed some degree of leakiness. Class II mutants (complementation groups B, C) were positive in viral-DNA synthesis with a small amount of capsid polypeptides production. Class I mutant (complementation group A) was an early mutant defective in viral-DNA synthesis but positive in induction of host-DNA synthesis. Transformation of Nil cells was observed with classes I and II mutants and not with class III mutants.Ten temperature-sensitive (ts) mutants were divided into seven complementation groups and into five classes by the available phenotypic criteria. Class V mutant (complementation group G) was positive in viral-DNA synthesis and capsid polypeptides production with extreme leakiness. Class IV mutants (complementation groups E, F) were positive in viral-DNA synthesis and capsid polypeptides production. Class III mutants (complementation groups C, D) were quite similar to class IV except for reduced hexon production. Class II mutants (complementation group B) were early mutants defective in viral-DNA synthesis but positive in induction of host-DNA synthesis. Class I mutants (complementation group A) were similar to class II but with a reduced degree of induction of host-DNA synthesis. Transformation of Nil cells was observed with classes II, III, and IV mutants and not with I and V mutants.In brief, the phenotypic characterization of hr and ts mutants in infection of hamster cells showed a good correlation between complementation grouping and the defective function. Transformation of Nil cells was observed with most groups of the mutants except for the apparently leaky late groups and one group of early mutants under the restrictive conditions.     

Isolation of mutants of CHO cells resistant to 6(p-hydroxyphenylazo)-uracil I. A novel BrdU cross-resistant phenotype

Three classes of mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been isolated from mutagenized cultures of CHO cells. One class of these mutants designated HPU ^R A exhibits a unique form of cross-resistance to bromodeoxyuridine in that it is resistant to this drug only in the presence of thymidine. The molecular basis of the BrdU resistance is unknown but does not appear to involve the known targets of the drug. An interesting feature of these mutants is that they give rise, at a high frequency, to a subpopulation of cells which are much more resistant to BrdU.     

Repair in Escherichia coli alkB mutants of abasic sites and 3-methyladenine residues in DNA

Escherichia coli alkB mutants are sensitive to methyl methanesulfonate and dimethylsulphate, and are defective in the processing of methylated DNA. The function of the AlkB protein has not been determined. Here, we show that alkB mutants are not defective in repairing several different types of potentially toxic DNA lesions that are known to be generated by MMS, including apyrimidinic and apurinic sites, and secondary lesions that could arise at these sites (DNA–protein cross-links and DNA interstrand cross-links). Also, alkB mutants were not sensitive to MeOSO₂-(CH₂)₂-Lex, a compound that alkylates the minor groove of DNA generating primarily 3-methyladenine.     

Hydrogen Peroxide Production from Glycerol Metabolism Is Dispensable for Virulence of Mycoplasma gallisepticum in the Tracheas of Chickens

Hydrogen peroxide (H₂O₂) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H₂O₂ production on virulence in vivo have never been assessed in any Mycoplasma species. To this end, we determined that the wild-type (WT) R_low strain of the avian pathogen Mycoplasma gallisepticum is capable of producing H₂O₂ when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely, glpO, glpK, and glpF, were identified. All mutants assessed were incapable of producing H₂O₂ and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H₂O₂ when grown in glycerol, while the naturally attenuated F strain does produce H₂O₂. Chickens were infected with one of two glpO mutants, a glpK mutant, R_low, or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, all glp mutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H₂O₂ production/cytotoxicity and virulence for this Mycoplasma species in its natural host. However, it is possible that glycerol metabolism is required by M. gallisepticum in a niche that we have yet to study.     

The sharp phase of respiratory inhibition during amino acid starvation in Escherichia coli is RelA-dependent and associated with regulation of ATP synthase activity

Amino acid starvation causes an RelA-dependent increase in the regulatory nucleotide (p)ppGpp that leads to pleiotropic changes in Escherichia coli metabolism, but the role of (p)ppGpp in regulation of respiration remains unclear. Here we demonstrate that amino acid starvation is accompanied by sharp RelA-dependent inhibition of respiration. The sharp phase of inhibition is absent in relA mutants, and can be prevented by translation inhibitors chloramphenicol and tetracycline, which abolish accumulation of (p)ppGpp. Single knockouts of any components of the respiratory chain do not affect inhibition of respiration. Studies of dO₂ changes in various atp mutants indicate that ATP synthase is probably the primary target of (p)ppGpp-mediated respiratory control. Inhibition of respiration induced by amino acid starvation is followed by transient perturbations in the membrane potential (Δψ) and K⁺ fluxes and leads to transient acceleration of superoxide production and H₂O₂ accumulation in the medium. High levels of H₂O₂ and superoxide formation and induced activity of antioxidant systems in the atpC mutant indicate the important role of ATP synthase in controlling the production of reactive oxygen species. The new function of (p)ppGpp, discovered here, expands the understanding of its role in metabolic reprogramming during the adaptive response to stresses.     

Restoration of the DNA damage resistance of Deinococcus radiodurans DNA polymerase mutants by Escherichia coli DNA polymerase I and Klenow fragment

Deinococcus radiodurans and other species of this genus share extreme resistance to ionizing radiation and many other agents that damage DNA. D. radiodurans mutant strains defective in a deinococcal DNA polymerase that is homologous with E. coli DNA polymerase I are highly sensitive to DNA damage. In the current work we have inquired whether E. coli DNA Pol I can substitute for D. radiodurans Pol in partially or fully restoring to pol⁻ D. radiodurans mutants the extreme DNA damage-resistance typical of this organism. The E. coli polA gene or a 5′-truncated polA gene that encodes the Klenow fragment were introduced and expressed in two different D. radiodurans pol⁻ mutants: Strain 303, which is a chemically mutagenized derivative, and strain 6RIA, which is isogenic with wild-type D. radiodurans except for an insertional mutation within the pol gene. Expression of E. coli polA in both of these mutants fully restored wild-type resistance to ionizing- and UV₂₅₄-radiation and mitomycin-C exposure. Expression of the Klenow fragment-encoding gene restored wild-type resistance to D. radiodurans strain 303, but only partial resistance to strain 6RIA. The observation that E. coli DNA Pol I is as effective as D. radiodurans Pol in restoring damage resistance, indicates that D. radiodurans DNA Pol per se does not have special properties that are essential or prerequisite for expression of the extreme resistance of D. radiodurans.     

Induction of interferon by temperature-sensitive mutants of Newcastle disease virus

Spontaneously-selected and mutagen-induced temperature-sensitive (ts) mutants of Newcastle disease virus (NDV) were used to study interferon induction in chick embryo (CE) cells at temperatures permissive (37°) and nonpermissive (42°) for virus replication. Both infectious and UV-irradiated virus were tested for interferon-inducing ability in cells pretreated or not pretreated with homologous interferon. At 37°, only UV-irradiated NDV was capable of inducing interferon in cells not treated with interferon before infection. In cells pretreated with interferon, on the other hand, both unirradiated and UV-irradiated virus stimulated the production of interferon. At 42°, the interferon-inducing phenotype for some UV-irradiated ts mutants was dependent on whether or not cells were pretreated with interferon. For example, out of 10 mutants examined, one UV-irradiated ts mutant induced interferon in both untreated and interferon pretreated cells; 7 mutants failed to induce in untreated cells but induced from 25–100% of the wild-type level of interferon in cells pretreated with interferon; and two mutants failed to induce interferon in both types of cells. In addition, one mutant (NDV₀ts-100) induced low or undetectable levels of interferon at both 37° and 42°, conditions under which wild-type virus (NDV₀) produced significant levels of interferon. Co-infection of cells with UV-irradiated ts-100 and a preparation of NDV₀ exposed to prolonged irradiation resulted in considerable production of interferon. These results suggest the possibility that more than one virus function may be involved in interferon induction by NDV in CE cells.     

Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus.

Four DNA-negative temperature-sensitive (ts) mutants of human cytomegalovirus, belonging to different complementation groups, were studied for their ability to induce cell DNA synthesis and DNA polymerase in permissive human embryo lung (HEL) and nonpermissive rabbit lung (RL) cells. These is mutants stimulated host cell DNA synthesis in HEL and RL cells and DNA polymerase activity in HEL and RL cells at permissive (33.5°) and nonpermissive temperatures (39.5°). Salt stimulation of induced DNA polymerase activity was used to distinguish between virus and cell DNA polymerase from HEL cells. DNA polymerase activity was stimulated by 100 mM (NH₄)₂SO₄ at either 33.5 or 39.5° in cultures infected with three of the mutants (ts 9, is 153, and ts 155). However, DNA polymerase activity was not stimulated by 100 mM (NH₄)₂SO₄, in cultures infected with one of the ts mutants (ts 13). These data suggest that at least four cistrons control the synthesis of virus DNA and that virus DNA synthesis is not required for the induction of cell DNA synthesis.     

Protease activation mutants of sendai virus. Activation of biological properties by specific proteases.

A new class of Sendai virus mutants (pa mutants) is described that exhibit altered specificities with respect to protease activation of infectivity and altered host range. Sendai virus requires proteolytic cleavage of a virion glycoprotein (F₀ to F) in order to be infective. Wild-type virus can be activated in vitro by treatment with trypsin, but not chymotrypsin or elastase, or in vivo by addition of trypsin to cells, e.g., MDBK, which lack activating protease. Mutants have been isolated that are activated by chymotrypsin (pa-c mutants) or elastase (pa-e mutants). Some mutants are no longer activated by trypsin, and these mutants have lost the ability to undergo multiple-cycle replication in the embryonated chick egg unless chymotrypsin or elastase is added to the mutants also activate hemolysis. These findings with pa mutants support the previous conclusion based on results with wild type virus, that a host-dependent cleavage of the F₀ protein is required for the infectivity of Sendai virus and for activation of hemolyzing and cell-fusing activities. The results obtained indicate that the host range and tissue tropism of Sendai virus are determined at least in part by the availability of the appropriate protease required for activation of infectivity.     

Mutations altering genetic recombination and repair of DNA in bacteriophage T4.

A search was made for sublethal mutants of bacteriophage T4 defective in genetic recombination. T4D⁺ was mutagenized with proflavin, and phage from minute plaques were tested for their sensitivity to ultraviolet (uv) irradiation. Nine uv-sensitive mutants were found, of which two exhibit decreased recombination. The mutations are located in a previously unknown T4 gene-gene w. Seven other minute-plaque-forming, uv sensitive mutants exhibit increased recombination; in each of these seven cases, the mutations were found to be in gene 58 (DNA-delay).The newly located gene w maps between genes 24 and 25. Mutants defective in gene w are sensitive to uv, to methyl methanesulfonate, and to hydroxyurea, although they degrade host DNA normally. Incorporation of [³H]thymidine into DNA is normal. A delayed and reduced phage yield appears to be caused by inefficient packaging of DNA in w mutants. This inefficient packaging appears to be caused by failure to form or to maintain a normal complement of concatemeric DNA.Intracellular DNA in 58^? infection was examined for unusual properties which could explain increased recombination. The normal rapidly sedimenting complex containing concatemeric DNA is present, but is formed more slowly than in wild-type infection. Joining of short DNA chains produced by discontinuous synthesis is normal, as is attachment of parental and newly synthesized DNA to a rapidly-sedimenting cell component (presumably membrane). Parental DNA is degraded, apparently by an exonuclease. Both the production of acid-soluble material from parental DNA and the increase in genetic recombination are eliminated by additional mutations in genes 46 and 47.Genetic evidence indicates that both 58 and w mutants are defective in the same uv repair pathway as the uv sensitive T4 mutants x and y; none of the double mutants is more sensitive than each of the respective single mutants. Some of these double mutants, however, have cumulative effects on recombination, indicating a more complicated interaction than a discrete unilinear pathway.The x and y strains currently available were found to contain additional mutations. The original x strain contains several mutations which affect plaque morphology. The original y strain, y₁₀, contains a mutation which affects plaque morphology, and the y₁₀₀ strain contains a mutation which causes the y defect to be lethal. The purified x and y strains make minute plaques and retain the reduced burst size, lowered recombination, uv sensitivity of the originally described mutants.     

Complementation between temperature-sensitive (ts) and host range nontransforming (hr-t) mutants of polyoma virus.

Two major classes of polyoma mutants are defective in cell transformation: early temperature-sensitive mutants of the tsA type which are defective in viral DNA synthesis and transformation at 39°, but not at 32°; and host range nontransforming (hr-t) mutants which fail to transform at either temperature. Mixed infection of mouse 3T3 cells by hr-t mutants and early tsA-type mutants results in enhanced growth of the tsA-type mutants at 39°, indicating that the hr-t mutants can supply the early viral function required for viral DNA synthesis. The hr-t mutants also complement late is mutants which fail to produce infectious progeny at 39° because of alterations in the 45,000-dalton major virion protein. Mixed infection of hamster BHK or rat Y1 cells by hr-t and tsA-type mutants results in efficient transformation at 39°, indicating that the two classes of mutants can complement for transformation. No complementation is observed in pair-wise crosses among the early tsA-type mutants alone. The tsA-type mutants are located in the distal portion of the early region of the polyoma genome [Miller, L. K., and Fried, M. (1976) J. Virol.18, 824–832]. The hr-t mutants are located in the proximal portion [Feunteun, J., Sompayrac, L., Fluck, M., and Benjamin, T. (1976) Proc. Nat. Acad. Sci. USA]. These results suggest that the early region of the polyoma genome is divided into two functional regions which can complement for transformation. The ts3 mutant of polyoma is located in the proximal portion of the late region.     

The role of residues in binding loop A in desflurane and propofol modulation of recombinant 5-HT3A receptor

5-Hydroxytryptamine type 3 (5-HT₃) receptor is modulated by general anesthetics and regarded as a possible site of anesthetic adverse action. Although two amino acids located in transmembrane (TM) 2 and TM3 of LGICs were reported as critical for allosteric modulation by anesthetics and alcohols, other residues could regulate anesthetic modulation. Earlier studies identified the role of glutamate 129 and phenylalanine 130 in the non-TM extracellular region in the agonist binding and coupling in the 5-HT_3A receptor. We investigated whether these non-TM amino acids are involved in desflurane and propofol modulation of the 5-HT_3A receptor in mutant 5-HT_3A receptors (mutants) expressed in Xenopus laevis oocytes. E129D and F130Y mutants were functionally expressed but E129Y and F130S mutants were not gated by serotonin. The wild type and F130Y mutants demonstrated positive modulation by desflurane at 6 and 12 vol.%. In contrast, E129D mutants were inhibited by desflurane in a concentration dependent manner. Propofol (1–100 μM) demonstrated depression of the currents in all receptors examined. These findings suggest the role of non-TM residues in the extracellular domain in the anesthetic modulation of the 5-HT_3A receptor.     

A Novel Class of Acetylcholinesterase,Revealed by Mutations,in the Nematode Caenorhabditis elegans

In ace-2;ace-1 double mutants of the nematode C. elegans, where the major acetylcholinesterase (AChE) classes A and B have been eliminated by mutation, the animals are viable and residual AChE activity remains. This residual activity differs markedly from AChE classes A and B; its K_m for acetylcholine is 1000-5000-fold lower, its resistance to eserine is 3000-260,000-fold higher, it is markedly more thermo-labile, and it can be separated from classes A and B by ion exchange chromatography. It has been designated class C AChE. Class C AChE of indistinguishable properties is also present in wild type C. elegans, at levels approximating those seen in ace-2;ace-1 double mutants, suggesting that it is controlled by a third, as yet unidentified, gene. Amongst other sources examined, class C-like AChE has been detected in another nematode species, Stepkanurus dentatus, but not in Drosophila melanogaster, Torpedo californica, or Rattus rattus.