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PubMed2000

Two classes of sir3 mutants enhance the sir1 mutant mating defect and abolish telomeric silencing in Saccharomyces cerevisiae.

Silent information regulators, or Sir proteins, play distinct roles in chromatin-mediated transcriptional control at the silent mating-type loci, telomeres, and within the rDNA repeats of Saccharomyces cerevisiae. An unusual collection of sir3 mutant alleles was identified in a genetic screen for enhancers of the sir1 mutant mating-defective phenotype. These sir3-eso mutants, like the sir1 mutant, exhibit little or no mating defects alone, but the sir1 sir3-eso double mutants are essentially nonmating. All of the sir3-eso mutants are defective in telomeric silencing. In some mutants, this phenotype is suppressed by tethering Sir1p to telomeres; other mutants are dominant for mating and telomeric silencing defects. Additionally, several sir3-eso mutants are nonmating in combination with the nat1 N-terminal acetyltransferase mutant. The temperature-sensitive allele sir3-8 has an eso phenotype at permissive temperature, yet acts as a null allele at restrictive temperature due to loss of sir3-8 protein. Sequence analysis showed that eight of the nine sir3-eso alleles have mutations within the N-terminal region that is highly similar to the DNA replication initiation protein Orc1p. Together, these data reveal modular domains for Sir3p and further define its function in silencing chromatin.

Alleles↗

PubMed1999

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.

Amino Acids↗

PubMed1982

Yeast mutant defective in synthesis of phosphatidylinositol. Isolation and characterization of a CDPdiacylglycerol--inositol 3-phosphatidyltransferase Km mutant.

1. A Km mutant of Saccharomyces cerevisiae with a lesion in CDPdiacylglycerol-inositol 3-phosphatidyltransferase was isolated. The mutant required a high concentration of myo-inositol for growth. 2. The CDPdiacylglycerol-inositol 3-phosphatidyltransferase in the mutant cells showed an apparent Km for myo-inositol over 200-times higher than that of the enzyme in wild-type cells. The maximum velocity of the mutant enzyme was comparable to that of the wild-type enzyme. 3. In mutant cells, labelled myo-inositol, phosphate and acetate were incorporated into phosphatidylinositol at much slower rates than in wild-type cells. The phosphatidylinositol content of mutant cells was markedly lower than that observed in wild-type cells. 4. Genetic analysis showed that the growth phenotype of the mutant arose from a single nuclear gene mutation in a gene coding for CDPdiacylglycerol-inositol 3-phosphatidyltransferase. 5. The mutant showed a normal level of phosphatidylserine synthase activity. The phosphatidylserine synthase gene was located between ura3 and hom3 on chromosome V, whereas the CDPdiacylglycerol-inositol 3-phosphatidyltransferase gene showed no linkage with ura3. 6. Labelled acetate was incorporated into various lipids including triacylglycerols, diacylglycerols, sterol esters and phospholipids other than phosphatidylinositol at faster rates in mutant cells than in wild-type cells. Incorporation into both the fatty acid and the sterol moieties was facilitated in the mutant. 7. A striking change in the cell-division process was observed when phosphatidylinositol synthesis was limited. The results showed that phosphatidylinositol synthesis is involved in the cell-division cycle of yeast.

CDP-Diacylglycerol-Inositol 3-Phosphatidyltransfer↗

PubMed1967

Analysis of sporulation mutants. I. Response of uracil incorporation to carbon sources, and other mutant properties.

Mutants deficient in sporulation were isolated and characterized with respect to antibiotic and protease activity, transformability, growth, and sporulation. All but two mutants could grow on minimal medium containing glucose. The inability of most mutants to incorporate uracil into trichloroacetic acid-precipitable material (ribonucleic acid) during the developmental period, and their response to a number of carbon sources, were used to characterize their biochemical blocks. Reproducible measurements of these responses were possible when the pH of the culture, which changed during growth and greatly influenced the rate of uracil uptake, was adjusted to 6.5. By their response to ribose and glutamate, the sporulation mutants could then be divided into four groups. All mutants of the first three groups produced antibiotic activity against Staphylococcus aureus, whereas all mutants, except one, of the fourth group produced none or very little of this activity. Mutants which did not respond to glutamate belonged to the first three groups; they also grew slowly or not at all on glutamate as sole carbon source. One of these mutants lacked succinic dehydrogenase activity. The results indicate that most of our sporulation mutants are unable to produce or utilize a natural carbon precursor, which is normally used as a slowly available carbon and energy source via the Krebs cycle when other carbon sources are used up. It enters the Krebs cycle as a precursor of alpha-ketoglutarate, probably via acetylcoenzyme A. All mutants of group four are blocked in this pathway before alpha-ketoglutarate.

Anti-Bacterial Agents↗

PubMed1988

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs.

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

Base Sequence↗

PubMed1990

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication.

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.

Animals↗

PubMed1985

Characterization of a cyclic AMP-resistant Chinese hamster ovary cell mutant containing both wild-type and mutant species of type I regulatory subunit of cyclic AMP-dependent protein kinase.

We have characterized a cyclic AMP-resistant Chinese hamster ovary (CHO) cell mutant in which one of two major species of type I regulatory subunit (RI) of cyclic AMP-dependent protein kinase is altered. Wild-type CHO cell extracts contain two cyclic AMP-dependent protein kinase activities. As shown by DEAE-cellulose chromatography, there is a peak of type I protein kinase activity in mutant extracts, but the type II protein kinase activity is considerably reduced even though free type II regulatory subunit (RII) is present. The type I kinase from the mutant has an altered RI (RI*) whose KD for the binding of 8-N3[32P] cAMP (KD = 1.3 X 10(-5) M) is increased by more than 200-fold compared to RI from the wild-type enzyme (KD = 5.5 X 10(-8) M). No differences were found between the catalytic subunits from the wild-type and mutant type I kinases. A large portion of RI in mutant and wild-type extracts is present in the free form. The RI* derived from mutant type I protein kinase shows altered labeling by 8-N3[32P]cAMP (KD = 1.3 X 10(-5) M) whereas the free RI from the mutant is labeled normally by the photoaffinity label (KD = 7.2 X 10(-8) M), suggesting that the RI* which binds to the catalytic subunit is functionally different from the free form of RI. The decreased amount of type II kinase activity in the mutant appears to be due to competition of RI* with RII for binding to the catalytic subunit. Translation of mRNA from wild-type CHO cells results in the synthesis of two different charge forms of RI, providing biochemical confirmation of two different species of RI in CHO cells. Additional biochemical evidence based on isoelectric focusing behavior of 8-N3[32P]cAMP-labeled RI species and [35S]methionine-labeled RI from mutant and wild-type extracts confirms the charge heterogeneity of RI species in CHO cells. These genetic and biochemical data taken together are consistent with the conclusion that there are at least two different species of RI present in CHO cells and that one of these species is altered in the mutant analyzed in this work.

Affinity Labels↗

PubMed1983

Isolation of drug resistant mutants of varicella-zoster virus: cross resistance of acyclovir resistant mutants with phosphonoacetic acid and bromodeoxyuridine.

Mutants of Varicella-Zoster Virus (VZV) which are resistant to phosphonoacetic acid (PAA), bromodeoxyuridine (BuDR), and acyclovir (ACV) were obtained by serial passages of VZV with increasing concentrations of these drugs. A PAA-resistant mutant and a BuDR-resistant mutant were found also to be resistant to ACV. Five of 8 ACV-resistant mutants acquired resistance to PAA, but none acquired resistance to BuDR. The BuDR-resistant mutant did not induce viral thymidine kinase (TK) activity, but all the ACV-resistant mutants selected in ACV showed viral TK activity which was suppressed with anti-VZV serum and had almost the same electrophoretic mobility as that of the parent strain on polyacrylamide gel electrophoresis in non-denaturing conditions. However, in competitive TK assay with ACV, 2 of 8 ACV-resistant mutants showed no change of phosphorylation of radioactive thymidine, while the other 6 showed decreased phosphorylation of radioactive thymidine. It was suggested that TK induced by the former 2 ACV-resistant mutants had lost affinity to ACV, and so the mutants could grow in the presence of ACV. Thus of the 8 ACV-resistant mutants selected in ACV, 2 were sensitive to PAA with altered TK activity, 5 were resistant to PAA with unaltered TK activity, and 1 was sensitive to PAA with unaltered TK activity, and may have altered DNA polymerase activity to ACV, retaining sensitivity to PAA. These results suggest that resistance of VZV to ACV results from alterations in the virus-specified TK or DNA polymerase, as demonstrated in HSV resistant to ACV.

Acyclovir↗

PubMed1985

Residual benzodiazepine (BZ) binding in the cortex of pcd mutant cerebella and qualitative BZ binding in the deep cerebellar nuclei of control and mutant mice: an autoradiographic study.

In mutant mice 'Purkinje cell degeneration' (pcd), there is an almost complete degeneration of Purkinje cells followed subsequently by a partial degeneration of granule cells. Recent neurochemical studies have revealed a 50% decrease in benzodiazepine (BZ) receptors in 45-day-old pcd mutants after degeneration of the Purkinje cells. At 300 days there is an 80% decrease in BZ receptors concomitant with granule cell losses. To determine the histological localization of these receptor changes this autoradiographic analysis was conducted. An in vitro autoradiographic technique was used to explore [3H]flunitrazepam binding. BZ receptors were found to be more concentrated in the molecular than the granular layer of mutant and control cerebellar cortices. There was, nonetheless, no statistically significant difference in grain counts between control and mutant mice in any layer. Substantial atrophy of cerebellar structures, particularly of the molecular layer, occurred in the mutant mice. It began even before 45 days of age but was extreme at 300 days. When the appropriate mathematical correction factor was introduced for the layer atrophy there was a 60% decrease in grain count in 45-day-old mutants in the molecular layer and a 84% decrease in 300-day-old mutants compared to controls. The initial decrease in total BZ receptors in the 45-day-old mutant animals is associated with a selective loss of Purkinje cells. The amount of receptor binding which persists in the 300-day-old mutants in the molecular layer would appear to reflect binding in the remaining parallel fibers from granule cells which remain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Development and degeneration of retina in rds mutant mice: effects of light on the rate of degeneration in albino and pigmented homozygous and heterozygous mutant and normal mice.

The effect of light on the rate of visual cell loss in mice afflicted by the rds (retinal degeneration slow) gene was analyzed by comparing the changes in the thickness of the outer nuclear layer. Visual cell loss in the pigmented, homozygous mutant mice, maintained in cyclic light, is slightly slower than in the albino mutant mice. In the pigmented mutant mice, exposed to constant light, and in the albino mutant mice, reared in darkness, rate of cell loss is not significantly altered. In the albino animals exposed to constant light, the rate of cell loss is faster in the homozygous mutant than in the normal, and intermediate in the heterozygous mutant retina. The accelerated cell loss in the mutant retina progresses from the centre to the periphery, and affects the rods earlier than the cones. This resembles the photic lesion in the normal retina but is unlike the genetic lesion in the mutant retina which appears to progress from the periphery to the centre and affects both rods and cones. It is concluded that the visual cells in the retina of rds mutant mice are more vulnerable to photic damage than those in the retina of normal mice.

Albinism↗

PubMed1988

A survey of neurological mutant mice. II. Lipid composition of myelinated tissue in possible myelin mutants.

The lipids of white matter and peripheral nerve from neurological mutant mice with possible myelin abnormalities were analyzed by thin-layer chromatography and quantitated by densitometry. Eight mutants had major abnormalities in the central nervous system (CNS) and/or peripheral nervous system (PNS) tissues examined (optic nerve, and trigeminal and sciatic nerves). In the optic nerve of axJ/axJ, there were increases of 20-30% in the levels of the major phospholipids; peripheral nerve was normal. In bc3J/bc3J CNS, the major phospholipids and cholesterol were increased by 25-40%; the PNS was normal. In myd/myd CNS, there were increases of about 20% in the levels of both forms of cerebrosides and in the major phospholipids; in the PNS the lipids were normal. ot/ot CNS had 20-40% reductions of all the glycolipids and minor alterations in some of the phospholipids and cholesterol; the PNS had 20% losses of both forms of cerebrosides. In the PNS of ji/ji, there were decreases of 10-40% among the glycolipids and of 15-25% in three of the major phospholipids; the CNS was virtually normal. In the PNS of dtJ/dtJ, vb/vb and wr/wr, almost all lipids were significantly decreased. The CNS of dtJ/dtJ and vb/vb were normal; wr/wr had minor reductions of certain glycolipids and phospholipids. Six mutants had relatively minor lipid abnormalities in their myelinated tissues. In cr/cr PNS, there were elevated levels of the cerebrosides and major phospholipids; the CNS was virtually normal. In db/db CNS and PNS, there were reduced levels of the nonhydroxy forms of cerebroside and sulfatide. The major change in htr/htr was the elevation of all the glycolipids in the CNS. In the CNS of Lc/+, nonhydroxy cerebroside was reduced. In shm/shm PNS, nonhydroxy sulfatide was elevated and there were small decreases in some of the phospholipids. wl/wl CNS showed decreases among most of the glycolipids. Mutants homozygous for du, mto, spa and tg had virtually normal lipid levels in both the optic and peripheral nerves. Cholesterol ester, lysophospholipids and other unusual lipid species were not detected in any of the mutants. The plasmalogen forms of ethanolamine and choline phosphatides were at normal levels in all mutants that otherwise had significant alterations among their lipids. Although many alterations in lipid composition were found in these mutants, the changes were moderate compared to the classical myelin mutants and indicate that none of the mutants are severely myelin-deficient.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Recognition of H-2Kb mutant target cells by Moloney virus-specific cytotoxic T lymphocytes from bm13 (H-2Db mutant) mice. I. Full recognition of Kbm11 by Kb-restricted CTL.

In C57BL/6 (B6, H-2b) mice, the secondary in vitro CTL response against Moloney leukemia virus is restricted and regulated by the H-2Db locus. B6.C-H- 2bm13 ( bm13 ) mice, however, carrying a mutation at the Db locus, show an increased H-2Kb-restricted CTL response without a demonstrable CTL component restricted by the mutant Dbm13 molecule (D----K shift). These purely Kb-restricted bm13 virus-specific CTL were incubated with a series of Kb mutant virus-infected target cells to study the effect of the mutations at the target cell level. Of six Kb-mutant virus-infected target cells tested, bm1 cells were not recognized and bm8 cells were recognized only marginally by bm13 virus-specific CTL, whereas bm3 , bm5 , bm6 , and bm11 cells were fully recognized. Thus, the bm3 , bm5 , bm6 , and bm11 Kb mutants fully share the relevant H-2K restriction specificities with H-2Kb, whereas the bm1 mutant totally and the bm8 mutant almost completely lack these specificities. This result differs markedly from the restriction site relationships among B6 and these Kb mutants in other antigenic systems. The most striking example concerns the bm11 mutant, which is fully recognized by Moloney-specific CTL, but not at all by Sendai, minor H (H-3.1, H-4.2), and sulfhydryl hapten-specific CTL. Monoclonal anti-H-2Kb antibody B8-3-24 inhibited virus-specific lysis by bm13 CTL of all Kb virus-infected mutant target cells to which this antibody binds. Lysis of bm5 and bm11 but not of bm3 target cells was inhibited, in line with the fact that B8-3-24 antibody does not bind bm3 . On the other hand, not only bm5 and bm11 but also bm3 virus-infected target cells blocked virus-specific lysis to the same extent as syngeneic bm13 target cells. Therefore, bm13 virus-specific CTL populations do not recognize the discrete cluster alteration in the Kbm3 molecule, as identified by antibody B8-3-24. The bm1 and the bm8 mutations, which have structural alterations in completely different sites of the Kb molecule, show complete or almost complete loss, respectively, of Kb-Moloney restriction sites. This finding supports the notion that these virus-specific CTL recognize conformational determinants rather than linear amino acid sequences.

Animals↗

PubMed1984

Recognition of H-2Kb mutant target cells by Moloney virus-specific cytotoxic T lymphocytes from bm13 (H-2Db-mutant) mice. II. Relationship of Kbm3 and Kbm11 in restriction specificities and allodeterminants.

We have studied the effect of mutations in H-2Kb on recognition of Moloney virus antigens by Kb-restricted Moloney virus-specific cytotoxic T lymphocytes (CTL) generated from H-2Db-mutant B6.C-H- 2bm13 ( bm13 ) mice. On the basis of the bm13 virus-specific recognition pattern of a series of Kb-mutant virus-infected (V+) cells, these Kb mutants could be divided into two groups that either shared the relevant H-2K restriction specificities with H-2Kb ( bm3 , bm5 , bm6 , and bm11 ) or lacked them completely (bm1) and/or almost completely ( bm8 ). In the study presented in this report, we concentrated on the alloreactive CTL included in the repertoire of bm13 Moloney-specific CTL. These CTL lysed noninfected (V-) allogeneic target cells of many H-2 types, including all Kb-mutant cells tested (bm1, bm3 , bm5 , bm6 , bm8 , and bm11 ). Recognition of V- cells of two Kb mutants, bm3 and bm11 , which share a common amino acid substitution at position 77 of the H-2K molecule with an additional change at position 89 in bm3 , was compared with recognition of the other Kb-mutant V- target cells in cold target inhibition studies. These studies showed that bm3 and bm11 cells share a determinant or determinants on the H-2K molecule recognized by alloreactive CTL among the Moloney virus-specific CTL population not present on the other Kb-mutant cells; lysis of both target cells was inhibited only by bm3 and bm11 V- cells. On the other hand, lysis of bm3 V+ target cells by bm13 virus-specific CTL was inhibited by all Kb-mutant V+ cells ( bm3 , bm5 , bm6 , and bm11 ) that were killed virus specifically by bm13 CTL. Thus, common determinants on the H-2K molecule of these Kb-mutant V+ cells are recognized by Kb-restricted Moloney virus-specific CTL. From the difference in inhibition pattern of the lytic reaction against bm3 V+ vs bm3 V- cells, we conclude that determinants on the same H-2K molecule recognized by Moloney virus-specific CTL and allocross -reactive effector cells included in the Moloney virus-specific population are not identical.

Animals↗

PubMed2000

Tumor-derived p53 mutant C174Y is a gain-of-function mutant which activates the fos promoter and enhances colony formation.

SV40 large T antigen-induced primitive neuroectodermal tumors of the rat provide a model system to study induction and progression of primitive neuroectodermal tumors at the molecular level. A cell line derived from such a tumor reproducibly gave rise to malignant derivatives that ceased large T-antigen expression but harbored a mutant p53 allele with a common mutation at Cys(174) to Tyr (C174Y). To determine whether this p53 mutation contributes to tumor progression, we analyzed mutant C174Y functionally. Co-transfection experiments in Saos-2 cells with mutant or wild-type p53 and reporter genes linked to various p53-responsive promoters revealed that mutant C174Y failed to transcriptionally transactivate the Mdm2, Waf1, Cyclin G and Bax promoters. Loss of transcriptional activation correlated with loss of DNA-binding activity. Moreover, mutant C174Y exhibited a dominant negative effect on co-expressed wild-type p53. The ability of mutant p53 to repress the viral RSV, LTR or SV40 early promoters or the cellular fos promoter was likewise impaired. In contrast, it showed even induction of the fos promoter. Consistent with these observations, mutant C174Y was non-functional in the suppression of Saos-2 cell growth and even conferred a growth advantage to the cells. Surprisingly, mutant C174Y was also impaired in nuclear transport, as revealed by immunofluorescence analyses. Taken together, our results demonstrate that mutant C174Y possesses features that can positively contribute to cancer progression.

Amino Acid Substitution↗

PubMed2002

Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library.

Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest. In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology. After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated. After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography. We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution. This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer. The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure-activity requirements for CV-N's antiviral properties.

Amino Acid Substitution↗

PubMed1991

A novel phenotype of an excision-repair mutant in Neurospora crassa: mutagen sensitivity of the mus-18 mutant is specific to UV.

A UV-sensitive mutant has been isolated from UV-mutagenized conidia of Neurospora crassa. The mutation responsible for the lesion was mapped in linkage group VL, proximal to the nucleolus organizer region. We designated the mutant mus-18. The sensitivity of the mus-18 mutant to UV-irradiation was not particularly high, being less than twice that of the wild-type strain. However, the frequency of mutations at the ad-3 loci induced by UV was extremely high even at low doses, under conditions where survival rates of mus-18 cells were almost identical to those of wild-type cells. Photo-reactivation of UV damage was normal in the mus-18 mutant. Sensitivity to other mutagens, such as gamma rays, 4-nitroquinoline-1-oxide, N-methyl-N'-nitro-N-nitrosoguanidine, mitomycin C and methyl methanesulfonate, was similar to that of the wild type. Fertility of the mus-18 mutant was normal in homozygous crosses. These results suggest that mus-18 is an excision-repair mutant. Measurement of endonuclease-sensitive sites (ESS) after liquid-holding recovery from UV damage revealed that ESS remained unrepaired for longer than 18 h in the mus-18 mutant, while most were eliminated within 6 h in wild-type cells and in other UV-sensitive mutants. This result suggests that mus-18 is defective in the incision step of dimer excision. The mus-18 mutant provides the first example of an excision-defective mutation in eukaryotes, which is specific to UV damage.

4-Nitroquinoline-1-oxide↗

PubMed1977

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.

Adenine Phosphoribosyltransferase↗

PubMed2006

Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression.

Mutations in the TP53 tumor suppressor gene are the most frequent genetic alteration in human cancers. These alterations are mostly missense point mutations that cluster in the DNA binding domain. There is growing evidence that many of these mutations generate mutant p53 proteins that have acquired new biochemical and biological properties. Through this gain of function activity, mutant p53 is believed to contribute to tumor malignancy. The purpose of our study was to explore mutant p53 as a target for novel anticancer treatments. To this aim, we inhibited mutant p53 expression by RNA interference in three different cancer cell lines endogenously expressing mutant p53 proteins, and evaluated the effects on the biological activities through which mutant p53 exerts gain of function. We found that depletion of mutant p53 reduces cell proliferation, in vitro and in vivo tumorigenicity, and resistance to anticancer drugs. Our results demonstrate that mutant p53 knocking down weakens the aggressiveness of human cancer cells, and provides further insight into the comprehension of mutant p53 gain of function activity in human tumor.

Animals↗