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Showing papers on "Mutant published in 1993"


Journal ArticleDOI
12 Feb 1993-Cell
TL;DR: A recessive Arabidopsis mutant, ctr1, that constitutively exhibits seedling and adult phenotypes observed in plants treated with the plant hormone ethylene is isolated and the DNA sequences of four mutant alleles were determined.

1,861 citations


Journal ArticleDOI
TL;DR: The results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved inAutophagy in yeast.

1,653 citations


Journal ArticleDOI
22 Oct 1993-Science
TL;DR: An early step in ethylene signal transduction in plants may involve transfer of phosphate as in prokaryotic two-component systems.
Abstract: Ethylene behaves as a hormone in plants, regulating such aspects of growth and development as fruit ripening, flower senescence, and abscission. Ethylene insensitivity is conferred by dominant mutations in the ETR1 gene early in the ethylene signal transduction pathway of Arabidopsis thaliana. The ETR1 gene was cloned by the method of chromosome walking. Each of the four known etr1 mutant alleles contains a missense mutation near the amino terminus of the predicted protein. Although the sequence of the amino-terminal half of the deduced ETR1 protein appears to be novel, the carboxyl-terminal half is similar in sequence to both components of the prokaryotic family of signal transducers known as the two-component systems. Thus, an early step in ethylene signal transduction in plants may involve transfer of phosphate as in prokaryotic two-component systems. The dominant etr1-1 mutant gene conferred ethylene insensitivity to wild-type Arabidopsis plants when introduced by transformation.

1,482 citations


Journal ArticleDOI
11 Nov 1993-Nature
TL;DR: The hy4 mutant5 is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway.
Abstract: Specific responses to blue light are found throughout the biological kingdom. These responses--which in higher plants include phototropism, inhibition of hypocotyl elongation, and stomatal opening--are in many cases thought to be mediated by flavin-type photoreceptors. But no such blue-light photoreceptor has yet been identified or isolated, although blue-light responses in plants were reported by Darwin over a century ago, long before the discovery of the now relatively well characterized red/far-red light photoreceptor, phytochrome. Here we describe the isolation of a gene corresponding to the HY4 locus of Arabidopsis thaliana. The hy4 mutant is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway. The HY4 gene, isolated by gene tagging, was shown to encode a protein with significant homology to microbial DNA photolyases. As photolyases are a rare class of flavoprotein that catalyse blue-light-dependent reactions, the protein encoded by HY4 has a structure consistent with that of a flavin-type blue-light photoreceptor.

1,129 citations


Journal ArticleDOI
TL;DR: It is reported here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB), the first mutations shown to lie in a plant photoreceptor gene.
Abstract: Phytochromes are a family of plant photoreceptors that mediate physiological and developmental responses to changes in red and far-red light conditions. In Arabidopsis, there are genes for at least five phytochrome proteins. These photoreceptors control such responses as germination, stem elongation, flowering, gene expression, and chloroplast and leaf development. However, it is not known which red light responses are controlled by which phytochrome species, or whether the different phytochromes have overlapping functions. We report here that previously described hy3 mutants have mutations in the gene coding for phytochrome B (PhyB). These are the first mutations shown to lie in a plant photoreceptor gene. A number of tissues are abnormally elongated in the hy3(phyB) mutants, including hypocotyls, stems, petioles, and root hairs. In addition, the mutants flower earlier than the wild type, and they accumulate less chlorophyll. PhyB thus controls Arabidopsis development at numerous stages and in multiple tissues.

935 citations


Journal ArticleDOI
TL;DR: These data demonstrate a gain of function associated with p53 mutations in addition to the loss of function shown previously to be associated with mutations in this tumour suppressor gene.
Abstract: We report that the expression of murine or human mutant p53 proteins in cells with no endogenous p53 proteins confers new or additional phenotypes upon these cells. Mutant p53 proteins expressed in cell lines lacking p53 resulted in either enhanced tumorigenic potential in nude mice ((10)3 cells) or enhanced plating efficiency in agar cell culture (human SAOS-2 cells). Also, mutant human p53 alleles, unlike the wild-type p53 protein, could also enhance the expression of a test gene regulated by the multi-drug resistance enhancer-promoter element. These data demonstrate a gain of function associated with p53 mutations in addition to the loss of function shown previously to be associated with mutations in this tumour suppressor gene.

896 citations


Journal ArticleDOI
TL;DR: The nucleotide sequences of thePB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells.
Abstract: The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.

859 citations


Journal ArticleDOI
22 Oct 1993-Cell
TL;DR: It is suggested that dysfunction of the mucosal immune system may underly the pathogenesis of some types of IBD in humans.

720 citations


Journal ArticleDOI
19 Nov 1993-Cell
TL;DR: The genetic mapping of a locus that strongly modifies tumor number in Min/+ animals is reported, and this gene, Mom-1 (Modifier of Min-1), maps to distal chromosome 4 and controls about 50% of genetic variation in tumorNumber in two intraspecific backcrosses.

663 citations


Journal ArticleDOI
01 Oct 1993-Neuron
TL;DR: Evidence is presented that apoptosis may be the final common pathway of the disease process linking genotype to phenotype, and DNA fragmentation by internucleosomal cleavage is a cardinal feature of apoptosis.

648 citations


Journal ArticleDOI
TL;DR: A single genetic locus, designated dot (for defect in organelle trafficking), restored wild‐type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome–lysosome fusion to mutants belonging to both phenotypic classes.
Abstract: Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome-lysosome fusion and association of host cell organelles with bacteria-containing phagosomes ('recruitment'). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome-lysosome fusion to mutants belonging to both phenotypic classes.

Journal ArticleDOI
TL;DR: Results show that expression of both mutant variants of the mouse p53 gene significantly increases the cellular resistance of a variety of hematopoietic cell lineages to gamma radiation, providing direct evidence that p53 mutations affect the cellular response to DNA damage.
Abstract: Mouse and human tumors of diverse origin frequently have somatically acquired mutations or rearrangements of the p53 gene, or they have lost one or both copies of the gene. Although wild-type p53 protein is believed to function as a tumor-suppressor gene, it is as yet unclear how p53 mutations lead to neoplastic development. Wild-type p53 has been postulated to play a role in DNA repair, suggesting that expression of mutant forms of p53 might alter cellular resistance to the DNA damage caused by gamma radiation. Moreover, p53 is thought to function as a cell cycle checkpoint after irradiation, also suggesting that mutant p53 might change the cellular proliferative response to radiation. We have used transgenic mice expressing one of two mutant alleles of p53 to test this prediction. Our results show that expression of both mutant variants of the mouse p53 gene significantly increases the cellular resistance of a variety of hematopoietic cell lineages to gamma radiation. These observations provide direct evidence that p53 mutations affect the cellular response to DNA damage, either by increasing DNA repair processes or, possibly, by increasing cellular tolerance to DNA damage. The association of p53 mutations with increased radioresistance suggests possible mechanisms through which alterations in the p53 gene might lead to oncogenic transformation.

Journal ArticleDOI
TL;DR: These findings suggest that lpr mice are able to express a very low level of the Fas antigen, and that an insertion of the ETn into an intron of a mammalian expression vector dramatically but not completely reduced the expression efficiency.
Abstract: The mouse lpr (lymphoproliferation) mutation carries a rearrangement in the chromosomal gene for the Fas antigen, which mediates apoptosis. Isolation and characterization of mouse Fas antigen chromosomal gene from wild-type and lpr mice indicated an insertion of an early transposable element (ETn) in intron 2 of the Fas antigen gene of lpr mice. Hybrid transcripts carrying the Fas antigen and ETn sequences were expressed in the thymus and liver of the mutant. This indicated that premature termination and aberrant splicing of the Fas antigen transcript caused by the insertion of the ETn in the intron are responsible for the lymphoproliferation and autoimmune phenotype of the mutant mouse. On the other hand, an insertion of the ETn into an intron of a mammalian expression vector dramatically but not completely reduced the expression efficiency. These findings suggest that lpr mice are able to express a very low level of the Fas antigen.

Journal ArticleDOI
TL;DR: A genetic analysis of root development in Arabidopsis thaliana has identified mutants that have abnormal morphogenesis, and genetic combinations of the four mutants have provided insight into the regulation of growth and cell shape duringArabidopsis root development.
Abstract: A genetic analysis of root development in Arabidopsis thaliana has identified mutants that have abnormal morphogenesis. Four of these root morphogenesis mutants show dramatic alterations in post-embryonic root development. The short-root mutation results in a change from indeterminate to determinate root growth and the loss of internal root cell layers. The cobra and lion's tail mutations cause abnormal root cell expansion which is conditional upon the rate of root growth. Expansion is greatest in the epidermal cells in cobra and in the stele cells in lion's tail. The sabre mutation causes abnormal cell expansion that is greatest in the root cortex cell layer and is independent of the root growth rate. The tissue-specific effects of these mutations were characterized with monoclonal antibodies and a transgenic marker line. Genetic combinations of the four mutants have provided insight into the regulation of growth and cell shape during Arabidopsis root development.

Journal ArticleDOI
08 Jul 1993-Nature
TL;DR: The AXR1 protein is highly diverged from previously characterized E1 enzymes, however, and lacks a key cysteine residue that is essential for E1 activity, which may define a new class of enzymes in the ubiquitin pathway or it may have a novel function in cellular regulation which is unrelated to Ubiquitin conjugation.
Abstract: The plant hormone auxin has a central role in many aspects of plant growth and development. By screening for mutants of Arabidopsis that are resistant to exogenous auxin, we have identified several genes that are required for normal auxin response. One of these genes, AXR1, is defined by recessive mutations that confer auxin resistance to the roots, rosettes and inflorescences of mutant plants. In addition, axr1 mutants display a variety of morphological defects that are consistent with a reduction in auxin sensitivity. Here we isolate the AXR1 gene using a map-based approach and report that AXR1 encodes a new protein with significant sequence similarity to the ubiquitin-activating enzyme E1. The AXR1 protein is highly diverged from previously characterized E1 enzymes, however, and lacks a key cysteine residue that is essential for E1 activity. AXR1 may therefore define a new class of enzymes in the ubiquitin pathway or it may have a novel function in cellular regulation which is unrelated to ubiquitin conjugation.

Journal ArticleDOI
12 Feb 1993-Cell
TL;DR: The analyses of TCR γ and δ genes in the mutant mice suggest that intracellular mechanisms acting at the level of DNA rearrangement play key roles in the differential δ and γ gene rearrangements and in the generation of the highly restricted junctional sequences during fetal thymic development.

Journal ArticleDOI
01 Dec 1993-Neuron
TL;DR: Analysis of the mutant in the olfactory bulb suggests that the mutant phenotype involves a defect in cell migration, possibly through specific loss of the polysialylated form of N-CAM-180, which is expressed in the migration pathway.

Journal ArticleDOI
TL;DR: Results from GA3 dose-response experiments suggest that GA3 and spy-1 interact in an additive manner, consistent with models in which the SPY gene product regulates a portion of the GA signal transduction pathway.
Abstract: Three independent recessive mutations at the SPINDLY (SPY) locus of Arabidopsis confer resistance to the gibberellin (GA) biosynthesis inhibitor paclobutrazol. Relative to wild type, spy mutants exhibit longer hypocotyls, leaves that are a lighter green color, increased stem elongation, early flowering, parthenocarpy, and partial male sterility. All of these phenotypes are also observed when wild-type Arabidopsis plants are repeatedly treated with gibberellin A3 (GA3). The spy-1 allele is partially epistatic to the ga1-2 mutation, which causes GA deficiency. In addition, the spy-1 mutation can simultaneously suppress the effects of the ga1-2 mutation and paclobutrazol treatment, which inhibit different steps in the GA biosynthesis pathway. This observation suggests that spy-1 activates a basal level of GA signal transduction that is independent of GA. Furthermore, results from GA3 dose-response experiments suggest that GA3 and spy-1 interact in an additive manner. These results are consistent with models in which the SPY gene product regulates a portion of the GA signal transduction pathway.

Journal ArticleDOI
TL;DR: Data demonstrate that the exchange of a single valine into methionine at position 715 in the AR promoters trans-activation not only by testicular but also by adrenal androgens and progesterone may have significance in the process controlling the progression of prostatic carcinoma.
Abstract: Structural changes of the androgen receptor (AR) may contribute to the development of resistance to endocrine therapy in prostatic carcinoma. We have isolated AR cDNA fragments from seven tumor specimens derived from patients with advanced metastatic prostatic tumors. In one specimen obtained from a patient who failed to respond to endocrine and cytotoxic therapy we have detected a point mutation in the hormone-binding domain of the receptor. This AR mutation is a guanine-to-adenine transition at nucleotide 2671 that leads to substitution of methionine for the wild type valine at position 715. It is a somatic mutation because it was not present in the AR genomic DNA fragments isolated from prostatic and testicular tissues of the same patient. The mutant AR was recreated in an expression vector and transiently expressed in COS-7 and CV-1 cells. Hormone-binding assays revealed that the mutant receptor does not differ from the wild type receptor in its ability to bind androgen. The dissociation constant for ...

Journal ArticleDOI
TL;DR: Both mutability on the DNA level and altered function of the mutant serine 249 p53 protein are responsible for the observed mutational hot spot in p53 in HCC from AFB1-contaminated areas.
Abstract: Approximately half of hepatocellular carcinoma (HCC) from regions in the world with high contamination of food with the mycotoxin aflatoxin B1 (AFB1) contain a mutation in codon 249 of the p53 tumor suppressor gene. The mutation almost exclusively consists of a G-->T transversion in the third position of this codon, resulting in the insertion of serine at position 249 in the mutant protein. To gain insight into the mechanism of formation of this striking mutational hot spot in hepatocarcinogenesis, we studied the mutagenesis of codons 247-250 of p53 by rat liver microsome-activated AFB1 in human HCC cells HepG2 by restriction fragment length polymorphism/polymerase chain reaction genotypic analysis. AFB1 preferentially induced the transversion of G-->T in the third position of codon 249. However, AFB1 also induced G-->T and C-->A transversions into adjacent codons, albeit at lower frequencies. Since the latter mutations are not observed in HCC it follows that both mutability on the DNA level and altered function of the mutant serine 249 p53 protein are responsible for the observed mutational hot spot in p53 in HCC from AFB1-contaminated areas. Our results are in agreement with an etiological role of AFB1 in hepatocarcinogenesis in regions of the world with AFB1-contaminated food.

Journal ArticleDOI
TL;DR: The function of PHYA might be highly specialized and restricted to certain phases of Arabidopsis development, and appears to play only a minor role in the regulation of hypocotyl elongation under natural conditions.
Abstract: Phytochrome, a red/far-red-light photoreceptor protein of plants, is encoded by a small gene family. Phytochrome A (PHYA), the product of the PHYA gene, is the predominant molecular species of phytochrome in etiolated tissue and has been best characterized biochemically. To define a role for PHYA, we isolated new mutants, designated fre1 (far-red elongated), in Arabidopsis thaliana that were specifically deficient in PHYA spectral activity and protein accumulation. These mutants were identified on the basis of their long hypocotyl phenotype under continuous far-red light. Although the fre1 mutants lacked the hypocotyl response to continuous far-red light, their responses to continuous white light and to end-of-day far-red-light treatments were normal. Thus, PHYA appears to play only a minor role in the regulation of hypocotyl elongation under natural conditions. In contrast, the fre1 mutation affected greening a fre1 mutant was less able than the wild type to deetiolate after growth in the dark. However, the potentiation effect of a red-light pulse on accumulation of chlorophyll was not changed significantly in the fre1 mutants. Thus, the function of PHYA might be highly specialized and restricted to certain phases of Arabidopsis development.

Journal ArticleDOI
TL;DR: Data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions.
Abstract: Virulence of the plant pathogen Erwinia carotovora subsp carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes Production of these exoenzymes is controlled by a global regulatory mechanism A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments LuxI controls bioluminescence in Vfischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone Ec subsp carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of Vfischeri This extracellular complementation can also be achieved by Ecoli harboring the luxI gene from Vfischeri or by adding the synthetic Vfischeri autoinducer Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions

Journal ArticleDOI
TL;DR: It is concluded that even in a uniform genetic background, stochastic variation in the expression of a developmental circuit can result in dramatic differences in phenotypic consequences.
Abstract: We derived mice that carry a targeted insertion of a neor gene in the int-2 (Fgf-3) proto-oncogene coding sequences. The mutation was found to be recessive and mice that were homozygous for the insertion did not often survive to adulthood. The mutant mice had defects in the development of the tail and inner ear that could be correlated with disruption of int-2 expression in the posterior primitive streak and hindbrain or otic vesicle. While the tail phenotype was 100% penetrant, we found that the inner ear phenotype had reduced penetrance and variable expressivity. The variable expressivity could not be attributed to variability in the genetic background of the mutant allele or to leaky expression from the mutant allele. Thus, we conclude that even in a uniform genetic background, stochastic variation in the expression of a developmental circuit can result in dramatic differences in phenotypic consequences.

Journal ArticleDOI
TL;DR: A specific role is demonstrated for dynamin and for GTP in the initial stages of receptor-mediated endocytosis in cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs.
Abstract: Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.

Journal ArticleDOI
TL;DR: Human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions and wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial Cells immortalization by adenovirus 5.
Abstract: Although rodent cells have been immortalized following transfection with a mutant p53 gene, the role of p53 in the immortalization of human cells is unknown. Therefore, human epithelial cell lines were examined for p53 mutations in exons 4-9 which include the evolutionarily conserved regions. A spontaneously immortalized skin keratinocyte cell line, HaCat, and three ras-transfected clones, have a p53 mutational spectrum that is typical of ultraviolet light induced mutations. A normal finite lifespan cell strain (184) and two benzo[a]pyrene immortalized mammary epithelial cell lines derived from 184 (184A1 and 184B5) contain wild type p53 sequences in exons 4-9, although elevated levels of nuclear p53 indicate an alteration in the stability of the normally transient protein. Wild type p53 was found in human bronchial, esophageal and hepatic epithelial cells immortalized by SV40 T antigen gene and human renal epithelial cells immortalized by adenovirus 5. BEAS-2B, an SV40 T antigen immortalized bronchial epithelial cell line and two subclones, have a germline polymorphism at codon 47. Inactivation of p53 by mechanisms such as mutation or complexing with proteins of DNA tumor viruses appears to be important in the immortalization of human epithelial cells.

Journal ArticleDOI
TL;DR: The properties and functions of wild‐type and mutant p53 will be compared and contrasted here and elsewhere within this thematic issue and the mechanisms of inactivation of p53 function will be discussed.
Abstract: Tumorigenesis is characterized by a series of genetic alterations in both dominant oncogenes and tumor suppressor genes. A hallmark of tumor suppressor genes is that both alleles are generally altered during transformation, which usually represents a loss of function phenotype. The p53 tumor suppressor gene is the most frequently affected gene detected in human cancer. There is now growing evidence suggesting that mutation of p53 may involve not only a loss of function of wild-type p53 activity but also a gain of function phenotype contributed by the mutant p53 protein. The study of the biological properties and functions of both wild-type and mutant p53 is central to our understanding of human cancer. These properties and functions of wild-type and mutant p53 will be compared and contrasted here and elsewhere within this thematic issue. In addition, the mechanisms of inactivation of p53 function, which include: 1) mutation, 2) inhibition by viral oncogene products, 3) inhibition by cellular regulators, and 4) alteration in subcellular localization, will be discussed.

Journal ArticleDOI
05 Nov 1993-Cell
TL;DR: It is shown that the SIR3 and SIR4 gene products have a sub-nuclear localization similar to the telomere-associated RAP1 protein, which is found primarily in foci at the nuclear periphery of fixed yeast spheroplasts.

Journal ArticleDOI
TL;DR: The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain theEBNA- 3A mutation indicate that EBNA -3A is essential or critical for lymphocyte growth transformation and that the EB NA-3C mutation has a partial dominant negative effect.
Abstract: Recombinant Epstein-Barr viruses (EBV) with a translation termination codon mutation inserted into the nuclear protein 3A (EBNA-3A) or 3C (EBNA-3C) open reading frame were generated by second-site homologous recombination. These mutant viruses were used to infect primary B lymphocytes to assess the requirement of EBNA-3A or -3C for growth transformation. The frequency of obtaining transformants infected with a wild-type EBNA-3A recombinant EBV was 10 to 15%. In contrast, the frequency of obtaining transformants infected with a mutant EBNA-3A recombinant EBV was only 1.4% (9 mutants in 627 transformants analyzed). Transformants infected with mutant EBNA-3A recombinant virus could be obtained only by coinfection with another transformation-defective EBV which provided wild-type EBNA-3A in trans. Cells infected with mutant EBNA-3A recombinant virus lost the EBNA-3A mutation with expansion of the culture. The decreased frequency of recovery of the EBNA-3A mutation, the requirement for transformation-defective EBV coinfection, and the inability to maintain the EBNA-3A mutation indicate that EBNA-3A is essential or critical for lymphocyte growth transformation and that the EBNA-3A mutation has a partial dominant negative effect. Five transformants infected with mutant EBNA-3C recombinant virus EBV were also identified and expanded. All five also required wild-type EBNA-3C in trans. Serial passage of the mutant recombinant virus into primary B lymphocytes resulted in transformants only when wild-type EBNA-3C was provided in trans by coinfection with a transformation-defective EBV carrying a wild-type EBNA-3C gene. A secondary recombinant virus in which the mutated EBNA-3C gene was replaced by wild-type EBNA-3C was able to transform B lymphocytes. Thus, EBNA-3C is also essential or critical for primary B-lymphocyte growth transformation.

Journal ArticleDOI
TL;DR: The results presented here emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.
Abstract: Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon-stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN-alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C-terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild-type cells completely restored the response to both IFN-alpha and -gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN-alpha response of all genes tested so far. It failed, however, to restore the IFN-gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN-alpha and -gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.

Journal ArticleDOI
TL;DR: The results clearly show that oxidative stress resistance and potential life span are correlated in this organism, and they suggest that the natural product of age-1 either directly or indirectly downregulates the activities of several other genes as a function of age.
Abstract: Mutations in the age-1 gene double both the mean and maximum life span of Caenorhabditis elegans. They also result in an age-specific increase of catalase and Cu/Zn superoxide dismutase activity levels. The higher superoxide dismutase activity levels in age-1 mutants confer hyperresistance to the superoxide-anion-generating drug paraquat. The rate of superoxide anion production by microsome fractions declines linearly with age in age-1(+) worms, but, after an initial decline, is stabilized at a higher level in senescent age-1 mutant nematodes. These results clearly show that oxidative stress resistance and potential life span are correlated in this organism, and they suggest that the natural product of age-1 either directly or indirectly downregulates the activities of several other genes as a function of age.