A dominant temperature-sensitive assembly mutant of adenovirus type 2.

An assembly negative temperature-sensitive mutant of Ad2, ts48 was shown to exert dominance over other ts mutants and wild-type virus during coinfection, by inhibiting virion assembly. Dominance was only expressed at the nonpermissive temperature.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type2.

Fourteen temperature-sensitive mutants of human adenovirus type2, which differed in their plaquing efficiencies at at the permissive and nonpermissive temperatures by 4 to 5 orders of magnitude, were isolated. These mutants, which could be assigned to seven complementation groups, were tested for their capacity to synthesize adenovirus DNA at the nonpermissive temperature. Three mutants in three different complementation groups proved deficient in viral DNA synthesis. The DNA-negative mutant H2ts206 complemented the DNA-negative mutants H5ts36 and H5ts125, whereas mutant H2ts201 complemented H5ts36 only. Among the DNA-negative mutants, H2ts206 synthesized the smallest amount of viral DNA at the nonpermissive temperature (39.5 C). Data obtained in temperature shift experiments indicated that a very early function was involved in temperature sensitivity. In keeping with this observation, early virus-specific mRNA was not detected in cells infected with H2ts206 and maintained at 39.5 C. Prolonged (52 h) incubation of cells infected with H2ts206 at the nonpermissive temperature led to the synthesis of a high-molecular-weight form of viral DNA.     

Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation.     

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.     

Characterization of a temperature-sensitive, hexon transport mutant of type 5 adenovirus.

Infection of KB cells at 39.5 degrees C with H5ts147, a temperature-sensitive (ts) mutant of type 5 adenovirus, resulted in the cytoplasmic accumulation of hexon antigen; all other virion proteins measured, however, were normally transported into the nucleus. Immunofluorescence techniques were used to study the intracellular location of viral proteins. Genetic studies revealed that H5ts147 was the single member of a nonoverlapping complementation group and occupied a unique locus on the adenovirus genetic map, distinct from mutants that failed to produce immunologically reactive hexons at 39.5 degrees C ("hexon-minus" mutants). Sedimentation studies of extracts of H5ts147-infected cells cultured and labeled at 39.5 degrees C revealed the production of 12S hexon capsomers (the native, trimeric structures), which were immunoprecipitable to the same extent as hexons synthesized in wild type (WT)-infected cells. In contrast, only 3.4S polypeptide chains were found in extracts of cells infected with the class of mutants unable to produce immunologically reactive hexon protein at 39.5 degrees C. Hexons synthesized in H5ts147-infected cells at 39.5 degrees C were capable of being assembled into virions, to the same extent as hexons synthesized in WT-infected cells, when the temperature was shifted down to the permissive temperature, 32 degrees C. Infectious virus production was initiated within 2 to 6 h after shift-down to 32 degrees C; de novo protein synthesis was required to allow this increase in viral titer. If ts147-infected cells were shifted up to 39.5 degrees C late in the viral multiplication cycle, viral production was arrested within 1 to 2 h. The kinetics of shutoff was similar to that of a WT-infected culture treated with cycloheximide at the time of shift-up. The P-VI nonvirion polypeptide, the precursor to virion protein VI, was unstable at 39.5 degrees C, whereas the hexon polypeptide was not degraded during the chase. It appears that there is a structural requirement for the transport of hexons into the nucleus more stringent than the acquisition of immunological reactivity and folding into the 12S form.     

Isolation and characterization of temperature-sensitive mutants of adenovirus type 7.

Fifty temperature-sensitive mutants, which replicate at 32 degrees C but not at 39.5 degrees C, were isolated after mutagenesis of the vaccine strain of adenovirus type 7 with hydroxylamine (mutation frequency of 9.0%) or nitrous acid (mutation frequency of 3.8%). Intratypic complementation analyses separated 46 of these mutants into seven groups. Intertypic complementation tests with temperature-sensitive mutants of adenovirus type 5 showed that the mutant in complementation group A failed to complement H5ts125 (a DNA-binding protein mutant), that mutants in group B and C did not complement adenovirus type 5 hexon mutants, and that none of the mutants was defective in fiber production. Further phenotypic characterization showed that at the nonpermissive temperature the mutant in group A failed to make immunologically reactive DNA-binding protein, mutants in groups B and C were defective in transport of trimeric hexons to the nucleus, mutants in groups D, E, and F assembled empty capsids, and mutants in group G assembled DNA-containing capsids as well as empty capsids. The mutants of the complementation groups were physically mapped by marker rescue, and the mutations were localized between the following map coordinates: groups B and C between 50.4 and 60.2 map units (m.u.), groups D and E between 29.6 and 36.7 m.u., and group G between 36.7 and 42.0 m.u. or 44.0 and 47.0 m.u. The mutant in group A proved to be a double mutant.     

Genetic analysis of adenovirus type 2. I. Isolation and genetic characterization of temperature-sensitive mutants.

Temperature-sensitive mutants which replicate normally at 33 C but poorly at 39 C were isolated from nitrosoguanidine- or nitrous acid-mutagenized adenovirus 2 by (i) testing the cytopathic effect or inclusion body-forming capacity of random plaque isolates, or (ii) reduced plaque enlargement upon shifting from 33 to 39 C. Thirty-six mutants were isolated with 33 C/39 C plaque ratios varying from 20 to 10-5. Some of these mutants could be arranged into 13 groups by the complementation test. By means of recombination analysis a provisional linear genetic map was constructed.     

Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.     

Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase

B?ck, August (Purdue University, Lafayette, Ind.), Lia Eidlic Faiman, and Frederick C. Neidhardt. Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase. J. Bacteriol. 92:1076-1082. 1966.-To test our conclusion that Escherichia coli mutant I-9 possesses a valyl soluble ribonucleic acid (sRNA) synthetase that functions in vivo at 30 C but not at 37 C, measurements were made by use of the periodate method, of the level of charged valyl sRNA in this strain. A shift of temperature from 30 to 40 C resulted in a rapid discharging of valyl sRNA coordinate with the cessation of protein synthesis; at the same time, other species of sRNA, such as those for leucine, became fully charged. Identical results were obtained with a derivative of I-9 with relaxed ribonucleic acid (RNA) control. When P1 phage were grown on wild cells and then used at low multiplicities of infection to transduce temperature-resistant growth into I-9, complete cotransduction of normal valyl sRNA synthetase occurred. By means of the interrupted-mating technique, the structural gene for valyl sRNA synthetase was located on the E. coli chromosome map and found to be near thr, one-fifth of the length of the chromosome removed from the structural genes for the isoleucine-valine biosynthetic enzymes. Therefore, (i) the major valyl sRNA synthetase activity of I-9 appears to be temperature-sensitive in vivo, (ii) relaxed amino acid control over RNA synthesis does not appear to be a consequence of a normal charging of sRNA with a substitute molecule, and (iii) one structural gene for valyl sRNA synthetase is located on the E. coli chromosome not closely linked to the cistrons for the valine-biosynthetic enzymes.     

Further characterization of a temperature-sensitive transformation mutant in Caenorhabditis elegans.

The temperature-sensitive sex transformer tra-2 (b202) II of the nematode Caenorhabditis elegans causes the transformation of genotypically hermaphrodite worms into phenotypic males and sterile intersexes at restrictive temperature. In this note, we show that the entire gonad structure is transformed and that oocyte development is autonomous of the form of the gonad and of the presence of a cellular sheath. Four oocyte-specific proteins are present in male intersexes that produce oocytes but are lacking in transformed males and hermaphrodite intersexes that do not produce oocytes.     

Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.     

Assembly intermediates among adenovirus type 5 temperature-sensitive mutants.

Temperature-sensitive mutants from three different complementation groups, ts5, ts19, and 6s58, have been shown to accumulate assembly intermediates at the restrictive temperature. The polypeptide composition of these intermediates is similar to that of the wild type, including the precursor polypeptides pVI, pVII, and pVIII. ts5 and ts19 also contained cleaved precursors, indicating assembly into defective virions. The increase of infectious virus after temperature shift-down of ts19 and ts58 was rapid when compared with that of ts24, which does not accumulate intermediates, suggesting that intermediates formed at nonpermissive temperature may be processed to mature virus. However, shift-down experiments reveal that only a fraction of the intermediates are utilized for virus assembly and that degradation of intermediates occurs at the restrictive temperature.     

Genetic analysis of adenovirus type 2. VIII. Physical locations of temperature-sensitive mutations.

Temperature-sensitive mutations of human adenoviruses can be physically located on the viral genome by determining the DNA structures of recombinants formed in genetic crosses between different members of the same subgroup. We have analyzed the DNA structures of many interserotypic recombinants formed in crosses between temperature-sensitive (ts) mutants of adenovirus type 2 and adenovirus type 5 with the restriction endonucleases BamHI, EcoRI, HindIII, and and Sma I. In this way, we have mapped the physical coordinates of adenovirus type 2 (Ad2) ts1, Ad2 ts3, Ad2 ts4, and Ad2 ts48, and refined the mapping of Ad5 ts1.     

Physiological and genetical studies on a mutant of Salmonella typhimurium which is temperature-sensitive for DNA synthesis

Summary The following evidence supports the view that a temperature-sensitive mutant of Salmonella typhimurium (11 G) is defective in DNA synthesis initiation: a) the increment in DNA synthesis at 38° is abolished by prior completion of rounds of replication at 25°. b) The extent of the increment at 38° is greatly increased by prior growth in the presence of a DNA synthesis inhibitor. c) Density gradient centrifugation demonstrates that the terminal region of the chromosomes is preferentially replicated at 38°. d) Preferential replication of the chromosome origins occurs at 25° after a period at 38°. The parental strain in the presence of a DNA synthesis inhibitor or the mutant at 38° (without inhibitor) show increased sensitivity to the detergent sodium deoxycholate, possibly due to a secondary effect of DNA synthesis inhibition on membrane composition. Strains of 11 G carrying episomes transfer the episomes very poorly at 38° suggesting a rôle for the chromosomal initiation apparatus in episome transfer. Continued cell division of 11 G with the production of DNA-less cells at 38° is not due to the presence of a rec mutation and no secondary mutation responsible for such division has been found. The lesion maps close to leu on the Salmonella chromosome.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Biochemical characterization of a fructokinase mutant of Rhizobium meliloti.

A double mutant strain (UR3) of Rhizobium meliloti L5-30 was isolated from a phosphoglucose isomerase mutant (UR1) on the basis of its resistance to fructose inhibition when grown on fructose-rich medium. UR3 lacked both phosphoglucose isomerase and fructokinase activity. A mutant strain (UR4) lacking only the fructokinase activity was derived from UR3; it grew on the same carbon sources as the parent strain, but not on fructose, mannitol, or sorbitol. A spontaneous revertant (UR5) of normal growth phenotype contained fructokinase activity. A fructose transport system was found in L5-30, UR4, and UR5 grown in arabinose-fructose minimal medium. No fructose uptake activity was detected when L5-30 and UR5 were grown on arabinose minimal medium, but this activity was present in strain UR4. Free fructose was concentrated intracellularly by UR4 > 200-fold above the external level. A partial transformation of fructose into mannitol and sorbitol was detected by enzymatic analysis of the uptake products. Polyol dehydrogenase activity was detected in UR4 grown in arabinose-fructose minimal medium. The induction pattern of polyol dehydrogenase activities in this strain might be due to slight intracellular fructose accumulation.     

Biochemical characterization of a high-palmitoleic acid Helianthus annuus mutant.

In the present work we carried out analytical and biochemical studies on a new high-n-7 monounsaturated fatty acid sunflower (Helianthus annuus L.) mutant. This new line, which has been selected by classical methods of breeding and mutagenesis, shows contents of unusual acyl chains up to 20% (12% of 16:1DELTA9, 5% of 16:2delta9,12 and 6% of 18:1delta11), whereas those fatty acids are found in negligible amounts in common sunflower cultivars. This characterization involved in vivo incubations with radiolabeled acetate and measurement of the last enzymes involved in the intraplastidial de novo fatty acid synthesis: beta-ketoacyl-ACP synthase II, stearoyl-ACP desaturase (EC 1.14.19.2) and acyl-ACP thioesterases (EC 3.1.2.14). Results indicated that the high-palmitoleic acid phenotype was associated with a concerted reduction in the fatty acid synthase II activity with respect to the control lines and an increase of stearoyl-ACP desaturase activity with respect to the high-palmitate mutant line.     

Purification and molecular characterization of adenovirus type 2 DNA-binding protein.

An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 mug of binding protein (95 to 99% homogeneity) starting with 2 X 10(9) infected cells. By omitting the Sephadex G-200 step, 400 to 600 mug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native stage as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.4S and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated, suggesting that the Ad2 DNA-binding protein does not have a typical globular protein structure.     

Human adenovirus 2 temperature-sensitive mutant 112 contains three mutations in the protein IIIa gene

The temperature-sensitive (ts) mutant 112 of human adenovirus 2 is defective in the late stage of virus maturation. The region of functional mutation has been localised by marker rescue. It was observed that the ts mutation can be rescued by the left-hand part of the wild-type gene (nucleotides 12,301-12,891). By nucleotide sequencing, two mutations, both C to T (at position 12,386 and 12,741), were found in this region. The first one, in the glycine 20 codon, is silent, whereas the second changes alanine 145 to valine. A third mutation, which changed C to A (nucleotide 13,613), was identified in the right-hand part of the gene, resulting in the replacement of alanine-436 by threonine.