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PubMed2004

Mutant prevention concentrations for single-step fluoroquinolone-resistant mutants of wild-type, efflux-positive, or ParC or GyrA mutation-containing Streptococcus pneumoniae isolates.

Three fluoroquinolone-susceptible and five fluoroquinolone-resistant (two with ParC Ser79Phe mutations, one with a GyrA Ser81Phe mutation, and two that were efflux positive) Streptococcus pneumoniae isolates were exposed to one, two, four, eight, and sixteen times the MICs of ciprofloxacin, gatifloxacin, gemifloxacin, levofloxacin, and moxifloxacin. Mutational frequencies were calculated at each multiple of the MIC for which growth was observed. Mutant prevention concentrations (MPCs) and the multiple of the MIC at the MPC (MP(MIC)) were evaluated. All resulting mutants were sequenced for quinolone resistance-determining region changes in GyrA and ParC and were evaluated for reserpine-sensitive efflux. The MPC order was generally ciprofloxacin > levofloxacin > gatifloxacin > moxifloxacin > gemifloxacin. The MP(MIC) order varied depending on the genetic constitution of the original isolates from which the mutants were generated. For those mutants created from fluoroquinolone-susceptible isolates (those that had wild-type ParC and GyrA and were efflux negative), the MP(MIC) order was ciprofloxacin = moxifloxacin > gemifloxacin > levofloxacin > gatifloxacin. The MP(MIC)s of each fluoroquinolone for mutants created from isolates with a ParC mutation (with wild-type GyrA and efflux negative) were similar. A similar occurrence was observed with the mutants created from the efflux-positive isolates (with wild-type ParC and GyrA). The MP(MIC) order for the mutants created from the isolate with a GyrA mutation (with wild-type ParC and efflux negative) was ciprofloxacin = gemifloxacin > levofloxacin = moxifloxacin > gatifloxacin. Gatifloxacin, levofloxacin, and moxifloxacin may be intrinsically more able to prevent the development of resistance by fluoroquinolone-susceptible isolates, isolates that are efflux positive, or isolates that carry a GyrA mutation. However, once a ParC mutation is present, the MPC increases dramatically for all fluoroquinolones.

Anti-Infective Agents↗
PubMed1979

Escherichia coli mutants incapable of supporting replication of F-like plasmids at high temperature: isolation and characterization of mafA and mafB mutants.

Mutants of Escherichia coli K-12 defective in replication of F-like plasmids at a high temperature (42 degrees C) were found among threonine-independent (Thr+) revertants of a threonine-requiring F' stain after localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Transduction experiments with phage P1 permitted us to divide these mutations into two classes with respect to man location; some mutations were located between thr and ara at about 0.8 min, very close to maf-1 reported previously (Wada et al., J. Mol. Biol. 108:25-41, 1976 and the others probably were located between leu and azi at about 1.8 min. The former class of mutants designated mafA exhibited the same plasmid specificity as maf-1; replication of plasmids F and ColVB trp, but not R386 or R222, were affected at a high temperature. By contrast, the latter mutants designated mafB were defective in replication of nay of these plasmids at a high temperature. When a culture of mafA mutants carrying an F' plasmid was transferred from 30 to 42 degrees C, the plasmid replication as determined by incorporation of [3H]thymidine into covalently closed circular F DNA was markedly inhibited. Under certain conditions, the temperature shift-up caused severe growth inhibition of the mutant cells. Examination of merodiploids (mafA/FmafA+) for plasmid maintenance suggested that the two mafA mutations tested (mafA23 and mafA36) were both dominant, at least partially, over the wild-type mafA+ allele. These properties of the mafA mutants, manifested at the restrictive temperature, are similar to those previously reported for the maf-1 mutant. Taken together with other evidence it is likely that these mutations affect either the same gene (mafA) or a set of closely linked genes, playing a specific role in autonomous plasmid replication in E. coli.

Chromosome Mapping↗
PubMed1994

DnaK mutants defective in ATPase activity are defective in negative regulation of the heat shock response: expression of mutant DnaK proteins results in filamentation.

Site-directed mutagenesis has previously been used to construct Escherichia coli dnaK mutants encoding proteins that are altered at the site of in vitro phosphorylation (J. S. McCarty and G. C. Walker, Proc. Natl. Acad. Sci. USA 88:9513-9517, 1991). These mutants are unable to autophosphorylate and are severely defective in ATP hydrolysis. These mutant dnaK genes were placed under the control of the lac promoter and were found not to complement the deficiencies of a delta dnaK mutant in negative regulation of the heat shock response. A decrease in the expression of DnaK and DnaJ below their normal levels at 30 degrees C was found to result in increased expression of GroEL. The implications of these results for DnaK's role in the negative regulation of the heat shock response are discussed. Evidence is also presented indicating the existence of a 70-kDa protein present in a delta dnaK52 mutant that cross-reacts with antibodies raised against DnaK. Derivatives of the dnaK+ E. coli strain MC4100 expressing the mutant DnaK proteins filamented severely at temperatures equal to or greater than 34 degrees C. In the dnaK+ E. coli strain W3110, expression of these mutant proteins caused extreme filamentation even at 30 degrees C. Together with other observations, these results suggest that DnaK may play a direct role in the septation pathway, perhaps via an interaction with FtsZ. Although delta dnaK52 derivatives of strain MC4100 filament extensively, a level of underexpression of DnaK and DnaJ that results in increased expression of the other heat shock proteins did not result in filamentation. The delta dnaK52 allele could be transduced successfully, at temperatures of up to 45 degrees C, into strains carrying a plasmid expressing dnaK+ dnaJ+, although the yield of transductants decreased above 37 degrees C. In contrast, with a strain that did not carry a plasmid expressing dnaK+ dnaJ+, the yield of delta dnaK52 transductants decreased extremely sharply between 39 and 40 degrees C, suggesting that DnaK and DnaJ play one or more roles critical for growth at temperatures of 40 degrees C or greater.

Adenosine Triphosphatases↗
PubMed1994

holE, the gene coding for the theta subunit of DNA polymerase III of Escherichia coli: characterization of a holE mutant and comparison with a dnaQ (epsilon-subunit) mutant.

DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an alpha epsilon theta heterotrimer that incorporates both a polymerase subunit (alpha; dnaE) and a proofreading subunit (epsilon; dnaQ). The role of theta is unknown. Here, we describe a null mutation of holE, the gene for theta. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting alpha which was identical to a suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the theta subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of epsilon mutants cannot be explained on the basis of interference with theta function.

DNA Polymerase III↗
PubMed1981

Genetic analysis of gene 1.2 of bacteriophage T7: isolation of a mutant of Escherichia coli unable to support the growth of T7 gene 1.2 mutants.

The product of gene 1.2 of bacteriophage T7 is not required for the growth of T7 in wild-type Escherichia coli since deletion mutants lacking the entire gene 1.2 grow normally (Studier et al., J. Mol. Biol. 135:917-937, 1979). By using a T7 strain lacking gene 1.2, we have isolated a mutant of E. coli that was unable to support the growth of both point and deletion mutants defective in gene 1.2. The mutation, optA1, was located at approximately 3.6 min on the E. coli linkage map in the interval between dapD and tonA; optA1 was 92% cotransducible with dapD. By using the optA1 mutant, we have isolated six gene 1.2 point mutants of T7, all of which mapped between positions 15 and 16 on the T7 genetic map. These mutations have also been characterized by DNA sequence analysis, E. coli optA1 cells infected with T7 gene 1.2 mutants were defective in T7 DNA replication; early RNA and protein synthesis proceeded normally. The defect in T7 DNA replication is manifested by a premature cessation of DNA synthesis and degradation of the newly synthesized DNA. The defect was not observed in E. coli opt+ cells infected with T7 gene 1.2 mutants or in E. coli optA1 cells infected with wild-type T7 phage.

Chromosome Mapping↗
PubMed1983

Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5.

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.

Adenoviruses, Human↗
PubMed2005

Diminished myofibril organization in mutant axolotl hearts transfected with site-directed mutants of sarcomeric tropomyosins.

We used a model lacking endogenous sarcomeric tropomyosin, the cardiac mutant of the Mexican axolotl, to examine the effect of mutant tropomyosins on sarcomeric myofibril formation. Previous studies have introduced wild-type mouse alpha-tropomyosin into mutant hearts in organ culture with subsequent for-mation of organized myofibrils. This study examines the predominant embry-onic axolotl TPM-4 type tropomyosin (TPM4alpha), containing a conservative re- placement of glutamic acid for aspartic acid at the clinically important 175 site. In this study, ATmC-3 (TPM4alpha) promoted formation of organized myofibrils in hearts without endogenous tropomyosin. Site-directed mutagenesis of 175 glutamic acid with 175 glutamine or 175 lysine was toxic to the formation of organized myofibrils in mutant hearts in the absence of endogenous tropo-myosin. Cationic liposome co-transfection of both wild-type tropomyosin and Glu175Gln.TPM4alpha cDNA formed organized myofibrils in mutant hearts. A construct with GFP.Glu175Gln.TPM4alpha cDNA was used to confirm expression of the mutant fusion protein. Mutation at the 175 site in TPM4alpha type or TPM1alpha (striated muscle isoform of the TPM1 gene) was sufficient to alter the protein such that organized myofibrils would not form in ventricles of mutant hearts without endogenous tropomyosin.

Ambystoma↗
PubMed1999

Transcriptional regulation of PDGF-A and TGF-beta by +KTS WT1 deletion mutants and a mutant mimicking Denys-Drash syndrome.

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are rare diseases caused by the mutations of Wilms tumor gene, WT1. The common denominator in these syndromes is a nephropathy which is manifested by early-onset proteinuria, nephrotic syndrome and end stage renal failure. Although these syndromes are genetic models of nephropathy and the mutations of WT1 gene are characterized in these patients the mechanism how mutations of WT1 gene affect the embryonic kidney adversely has not been elucidated. Recently, there was a report that FS is caused by mutations in the donor splice site of WT1. These mutations predicted loss of +KTS isoform, which is one of the four splicing variants of WT1. In this study, two +KTS deletion mutants of WT1 were made as well as a WT1 mutant mimicking a mutation found in a patient who had diffuse mesangial sclerosis, end stage renal failure and Wilms tumor. Mutant embryonic kidney cell lines were established by transfection of 293 embryonic kidney cells with WT1 mutants. We investigated the transcription regulation of mutant WT1 among these cell lines using the reporter vectors containing PDGF-A and TGF-beta promoter sequence. Our results showed that the promoter activity of PDGF-A and TGF-beta, which are related to the progression of glomerular diseases, was modestly increased in the mutant cell mimicking the patent, while those activities were markedly increased in other two deletion mutant cell lines. This study demonstrated that +KTS WT1 mutation found in DDS affected the cytokine expression adversely in vitro. From these results, we suggest that the alteration of +KTS WT1 expression be responsible for the rapid progression of renal diseases in DDS and FS.

Child, Preschool↗
PubMed1978

Comparative susceptibility to amylases of starch granules of several single endosperm mutants representative of floury-opaque, starch-deficient, and modified starch types and their double-mutant combinations with opaque-2 in four inbred lines of maize.

Starch granules were prepared from kernels of eight single endosperm mutants, brittle-1, (bt1), brittle-2 (bt2), floury-1, floury-2, soft starch, opaque-1 (o1), shrunken-2 (sh2), and sugary-2 (su2), and their double-mutant combinations with opaque-2 (o2) of four inbred lines of maize (Zea mays L.), B37, C103, Oh43 and W64A. We compared the susceptibility of various starch granules to Rhizopus glucoamylase and pancreatin. Starch granules of the su2 and su2o2 mutants were digested by amylases much faster than those of the normal counterparts. Starch granules of the bt1, bt2, o1 and sh2 mutants tended to be digested by amylases faster than those of normal maize. Starch granules of double-mutant combinations with the o2 gene were, in general, digested to an extent very comparable to their respective non-opaque single mutant counterparts in each of their four inbred backgrounds. We followed the relative digestion of starch granules by using scanning electron microscopy. Starch granules of endosperm mutants susceptible to amylases showed numerous pin holes on the surface layer and the pores penetrated into the inner layers of the granules during the attack by amylases. In some of the granules the inner portion, which appeared terraced or step-shaped, could be seen. This may be indicative of layered internal structures of the granules.

Amylases↗
PubMed1989

Conformational stability and activity of ribonuclease T1 and mutants. Gln25----Lys, Glu58----Ala, and the double mutant.

Ribonuclease T1 (RNase T1) and mutants Gln25----Lys, Glu58----Ala, and the double mutant were prepared from a chemically synthesized gene, cloned and expressed in Escherichia coli. The wild-type RNase T1 prepared from the cloned gene was identical in every functional and physical property examined to RNase T1 prepared from Aspergillus oryzae. Urea and thermal unfolding experiments show that Gln25----Lys is 0.9 kcal/mol more stable and Glu58----Ala is 0.8 kcal/mol less stable than wild-type RNase T1. In the double mutant, these contributions cancel and the stability does not differ significantly from that of wild-type RNase T1. For the double mutant, the dependence of delta G on urea concentration is significantly greater than for wild-type RNase T1 or the single mutants. This suggests that the double mutant unfolds more completely in urea than the other proteins. The activity of Gln25----Lys is identical with that of wild-type RNase T1. The activities of Glu58----Ala and the double mutant are 7% of wild-type when GpC hydrolysis is measured (due to a 35-fold decrease in kcat), and 37% of wild-type when RNA hydrolysis is measured. Thus, Glu58 is important, but not essential to the activity of RNase T1.

Aspergillus oryzae↗
PubMed1983

[Transmission of amber mutants in bacteriophage T4. II. Thermal sensitivity of the multiplication of gene 26 amber mutants in Escherichia coli B cells and the absence of such sensitivity in the case of gene 33].

The dependence of multiplication of genes 26 and 33 amber mutants of T4D phage in a non-permissive host on the temperature in the range of 25-41 degrees C has been studied. The secondary mutations observed in the genome of gene 26 mutants amS105 and amNG114 were excluded by crossing these mutants with the wild type phage. It was established that multiplication of gene 26 amber mutants amS105x5, amN131 and amNG114x1 depends, essentially, on the temperature: the burst size of phage gradually decreased by about three orders, when the incubation temperature was changed from 25 degrees C to 41 degrees C. In the case of gene 33 amber mutants, their multiplication was only slightly dependent on the temperature, maximum burst size having been observed at 37 degrees C. The decrease in the burst size at 41 degrees C of gene 33 mutant amc18 was shown to be caused by secondary ts mutation in the phage genome. It is concluded that the phenomenon of the temperature sensitivity of multiplication of T4D phage amber mutants is gene specific.

Crosses, Genetic↗
PubMed1982

Repair of mitochondrial DNA in Saccharomyces cerevisiae. Induction of cytoplasmic petite mutants in a nuclear mutant exhibiting thermosensitive mitochondrial deoxyribonuclease activity.

Four nuclear thermosensitive mutants have been obtained in which induction of up 100% cytoplasmic petite mutants (rho-) is observed upon cell incubation at 36 degrees C. For a given incubation time at 36 degrees C, the percentage of rho- is increased by preliminary gamma-ray irradiation. Under these conditions, the induction of rho- is a linear function of the irradiation dose. The retention of genetic information by rho- and of mitochondrial DNA synthesis in vivo and in vitro exclude that the mutants are deficient in the replication of mitochondrial DNA. The degradation of mitochondrial DNA labeled with [3H]dTTP in isolated mitochondria, has been monitored at 26 degrees C and at 36 degrees C after addition of 0.5% Triton X-100 in the presence or in the absence of ethidium bromide. In assays carried out at 26 degrees C, the degradation of mitochondrial DNA is similar in the parental strain and in the mutant gamma s rho 2. However, at 36 degrees C, the degradation of mitochondrial DNA is slower in the mutant. We have shown that a mitochondrial membrane deoxyribonuclease acting on double-stranded DNA at acid pH is thermosensitive in the mutant. Analysis of the meiotic segregants of a tetrad issued from the cross of the mutant with an isogenic parental strain shows co-segregation of rho- induction and of nuclease thermosensitivity in a 2:2 Mendelian pattern. These results suggest that a mitochondrial deoxyribonuclease is involved in the repair of damages caused to mitochondrial DNA by elevated temperature and by x-rays.

DNA Repair↗
PubMed1994

Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992) J. Biol. Chem. 267, 5272-5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway-defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine-inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan(EthN-P-->)ManGlcN- and EthN-P-6Man(EthN-P-6)Man(EthN- P-->)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D-glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.

Animals↗
PubMed1994

Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide.

We describe a system that allows us to easily isolate and characterize mutants in yeast topoisomerase II that are resistant to antitumor agents that target this enzyme. The system uses yeast strains that are sensitive to those agents and that carry temperature-sensitive top2 mutations. The temperature-sensitive mutation allows the isolation of recessive drug-resistant mutations. The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity. We describe three mutants that we isolated using this system. Two of the three mutants show resistance to etoposide and amsacrine, while the third mutant is partially resistant to etoposide and fluoroquinolones but not to amsacrine. DNA sequence changes have been identified in all of these mutant TOP2 genes. The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser83 of E. coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria. One of the alleles that confers resistance to both etoposide and amsacrine, top2-103, has changes in amino acid 824 and amino acid 1186 of TOP2. Reconstruction of the mutations by oligonucleotide-directed mutagenesis demonstrates that the change at amino acid 824 is responsible for the drug resistance of this allele.

Amino Acid Sequence↗
PubMed1983

UV- and X-ray-sensitive double mutants of mouse L5178Y cells are synergistically more sensitive to 4-nitroquinoline-1-oxide than is either of the single mutants.

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.

4-Nitroquinoline-1-oxide↗
PubMed1997

Differences in the retinal GABA system among control, spastic mutant and retinal degeneration mutant mice.

Immunocytochemical methods were used to compare the GABA system in control mice and two mutant strains: spastic which has reduced glycine receptors and retinal degeneration mutant in which the photoreceptors degenerate and reportedly have increased GABA and GAD levels. We found that the spastic mutant retina had reduced GABA-immunoreactivity (IR) in the proximal retina, reduced staining for GAD-1440 in the OPL, and reduced GABAA receptor staining in the OPL, compared to control. The retinal degeneration mutant retinas had enhanced GABA-IR throughout the retina, particularly in Müller cells, bipolar cells and IPL, and enhancement of GABAA receptor staining in the OPL, compared to control. The distributions of GABA-IR, GAD-1440-IR and GABAA receptor-IR in retinas of spastic mutant mice that also expressed the retinal degeneration phenotype resembled those found in retinas of mice that expressed only the retinal degeneration phenotype rather than those that expressed only the spastic mutation. No differences were observed among the conditions for GAD-65, GAD-67 or GABA-T. Our results with the spastic and retinal degeneration mutant mice demonstrate that attenuation in the glycinergic system and photoreceptor degeneration, respectively, is accompanied by alterations in different aspects of the GABA system, giving impetus for caution in the interpretation of experiments involving genetic manipulation of complex phenotypes.

Animals↗
PubMed1989

Functional rescue of temperature-sensitive defects in the cell-cycle mutants of rat 3Y1 fibroblasts by the small t-antigen deletion mutant of simian virus 40.

We examined the effects of large T antigen of simian virus 40 (SV40) on the proliferation phenotypes of temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which cease proliferating in the G1 phase of the cell cycle at a restrictive temperature (39.8 degrees C). Four ts mutants, each representing independent complementation groups, were transformed with the dl-884 mutant of SV40 which lacks the unique coding region for small t antigen. In the case of two ts mutants, their transformed derivatives did not cease proliferation at 39.8 degrees C. In the other two mutants, the transformed cells continued to enter the S phase but the cells became detached from the dishes thereafter, at 39.8 degrees C. The proliferation phenotypes of the dl-884-transformed cells at 39.8 degrees C were quite similar with those of the same mutants transformed with the wild-type SV40. These results indicate that large T antigen alone is sufficient to overcome the inhibition of cellular entry into S phase caused by four different ts defects and determines the proliferation phenotypes of the cells after entering the S phase at a restrictive temperature, and that small t antigen does not alter the cellular phenotypes determined by large T antigen.

Animals↗