A strategy for quickly identifying all unique two-hybrid or library plasmids within a pool of yeast transformants.
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ATP-Dependent Proteases↗
Biomedical subjects
G R Stark
Publications and source records attributed to G R Stark.
Simian virus 40 large tumor antigen alone or two cooperating oncogenes convert REF52 cells to a state permissive for gene amplification.
Gene amplification is characteristic of tumors and continuous cell lines but not of primary, normal, diploid, senescing cells. However, the rat cell line REF52, which resembles primary cells in requiring expression of cooperating oncogenes for transformation, is unusual among cell lines as it is not permissive for amplification. REF52 cells did not form colonies in N-(phosphonacetyl)-L-aspartate (PALA), a drug for which the only known mechanism of resistance is amplification of the carbamoylphosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) gene. Colonies did form in a low concentration of methotrexate but did not contain amplified dihydrofolate reductase genes. Expression of two cooperating oncogenes in REF52 cells converted them to a state permissive for amplification. Cells expressing only the 12S E1A mRNA of adenovirus 5 did not give rise to PALA-resistant colonies, but expression of an activated ras gene together with E1A readily allowed the cells to form resistant colonies in which the CAD gene was amplified. Cells expressing E1A plus ras were fully transformed, but expression of simian virus 40 large tumor antigen alone converted REF52 cells to a state permissive for amplification without transforming them fully. The ability to manipulate gene amplification in REF52 cells by expression of oncogenes should contribute to an understanding of the nature of the permissive state.
Adenovirus Early Proteins↗
A protein tyrosine kinase in the interferon alpha/beta signaling pathway.
The mutant human cell line 11.1 is unresponsive to interferon alpha. Here we describe the genetic complementation of this mutant and the identification and cloning of the wild-type gene that corrects the defect. Using transfection with genomic DNA in conjunction with a powerful back-selection, we isolated a cosmid that reverts the mutant phenotype of 11.1 cells. The cosmid encodes a single message whose level is greatly reduced in mutant cells. Complementary DNAs were cloned and found to be virtually identical to tyk2, a human mRNA encoding a non-receptor protein tyrosine kinase of previously unknown function. This finding shows that tyk2 links the interferon alpha/beta receptor to the cytoplasmic transcription factor that mediates activation of interferon-responsive genes.
Amino Acid Sequence↗
Fusions near telomeres occur very early in the amplification of CAD genes in Syrian hamster cells.
Previous analyses by fluorescence in situ hybridization of structures present 20-30 cell generations after the primary events of mammalian gene amplification have shown that tens of megabases of DNA separate each copy of the selected gene in chromosomal arrays that contain up to 15 copies. Since these structures are very unstable, it is necessary to study amplified DNA as soon as possible after it has been formed to relate the structures observed to the primary mechanisms that generated them. Previously, new amplifications of the CAD gene were analyzed in colonies of 10(5) N-(phosphonoacetyl)-L-aspartate-resistant Syrian hamster BHK cells. CAD is on the p arm of chromosome B9 and the amplified genes were usually found in large extensions of B9p, with one copy in its normal position. We now report that dividing drug-resistant cells have been physically separated from static drug-sensitive cells, to allow the amplified structures to be observed only a few cell generations after they have been formed. The most informative results are that about one-third of the newly formed chromosomes carrying amplified CAD genes are dicentric and that about half of these carry two B9q arms. These observations reveal that recombination between the p telomeric regions of two B9 sister chromatids is an important primary event of amplification in this system. The resulting dicentric chromosomes can then enter bridge-breakage-fusion cycles that provide the means to increase the number of CAD genes per cell in successive generations by an asymmetric distribution at each cell division.
Animals↗
Induction of gene amplification by 5-aza-2'-deoxycytidine.
Treatment of Syrian hamster kidney cells with the demethylating agent 5-aza-2'-deoxycytidine (azadC) increased both the frequency and the rate of gene amplification appreciably. AzadC caused substantial DNA demethylation, which is likely to be responsible. The magnitude of the increases depended on the concentrations of both azadC and the drug used for selection. A transient stress response is not responsible since the increases were not dependent on cytotoxicity and were still apparent after several weeks. We discuss mechanisms by which azadC treatment may induce amplification by rendering DNA more prone to this process or by increasing the transcription of genes whose protein products stimulate amplification.
Animals↗
Interferon-dependent signaling pathways: DNA elements, transcription factors, mutations, and effects of viral proteins.
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DNA Transposable Elements↗
The antiviral effects of the interferons and their inhibition.
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Adenovirus E1A Proteins↗
High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons.
2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive mutants were isolated previously at a low frequency (ca. 10(-8)) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-alpha. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, greater than 3 x 10(-7). Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-alpha have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U3 and U4 are unresponsive to IFN-alpha and, surprisingly, also unresponsive to IFN-gamma. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and double-stranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U3A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (alpha/beta) IFNs. Band-shift complementation assays show that U3A contains the E gamma subunit but does not contain an active E alpha subunit after treatment with IFN-alpha.
Aminacrine↗
Inhibition of the cellular response to interferons by products of the adenovirus type 5 E1A oncogene.
Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.
Adenovirus Early Proteins↗
The control of interferon-inducible gene expression.
The alpha beta interferons (IFNs) transiently induce genes through an IFN-stimulable DNA response element (ISRE). IFN-cell surface receptor interaction triggers the cytoplasmic activation of the complex primary transcription factor E, which on translocation and interaction with the ISRE initiates transcription. Whether E is activated directly through the receptor(s) or through a more classical second message pathway(s) and the roles of additional factors in the alpha beta and gamma IFN responses remain to be established. Meanwhile analysis of mutants has revealed complexity and overlap in the alpha, beta and gamma IFN response pathways and the products of at least two viruses have been shown to inhibit IFN-inducible gene expression.
Base Sequence↗
Molecular analysis of a human interferon-inducible gene family.
Three functional members of the 1-8 gene family have been isolated on a single human genomic DNA fragment of less than 18 kb. The 1-8U and 1-8D genes are extremely similar; each is contained within a less than 2-kb fragment, has in its 5'flanking region two adjacent 14-base-pair sequences showing high similarity to interferon-stimulable response elements (ISREs) and has two highly related exons. The third gene (9-27) has a similar overall structure, shows substantial similarity to the 1-8s but has only one ISRE which is 3' of two CCAAT boxes not present in the 1-8U and D genes. The cDNA corresponding to the three genes share 120 nucleotides of identical sequence and show greater than 90% identity over 70% of the coding sequence. For the 1-8U and D genes the high similarity extends into the 5' non-coding and flanking regions. The open reading frames encode polypeptides that are likely to be of very similar structure. Antiserum to a conserved peptide detects a polypeptide(s) of about 14 kDa on PAGE which separates into three components on isoelectric focussing. The 9-27 and 1-8U genes are highly interferon-inducible the 1-8D gene is much less so. These differences are mimicked by the activities of the corresponding ISREs placed 5' of a marker gene in expression constructs. They presumably reflect differences in the interaction of the ISREs with the various interferon-inducible and constitutive factors that govern the interferon response.
Amino Acid Sequence↗
Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA.
Constructs expressing the core, surface, X, or polymerase proteins of hepatitis B virus were transfected into human cells. In transient assays, only the polymerase inhibited the responses to interferons alpha and gamma (IFN-alpha and -gamma). Stable expression of the polymerase was achieved in the cell line 2fTGH, which carries an IFN-inducible marker gene, by growth under conditions that select for inhibition of the response to IFN-alpha, but the clones grew poorly. When expressed alone, the terminal protein domain of the polymerase gene inhibited the response to IFN-alpha and the reverse transcriptase plus RNase H domains appeared to be toxic. Clones of cells expressing terminal protein alone, selected for the loss of response to IFN-alpha, grew normally and had no detectable response to IFN-alpha, IFN-gamma, or double-stranded RNA. Binding of IFN-alpha to these cells was not impaired but did not lead to activation of the E alpha subunit of the IFN-induced transcription factor E. These observations are of potential importance in relation to the pathogenesis of chronic hepatitis B virus infection and the resistance of such infection to IFN-alpha therapy.
Cell Line↗
Differential response of the human 6-16 and 9-27 genes to alpha and gamma interferons.
9-27 mRNA is expressed to a high level in response to both alpha and gamma interferons. In contrast, 6-16 mRNA is expressed well in response to alpha but very poorly in response to gamma interferon in human cells. The factors governing these different levels of expression were investigated. For both genes the major effect of both interferons is on transcription. A transcriptional bias in the 6-16 promoter/enhancer accounts in large part for the differential response of 6-16 to the two interferons. No single DNA element appears responsible; the smaller the 5' region analysed the lower the absolute activity and the smaller the differential response to alpha and gamma interferons observed. Both the 6-16 and 9-27 mRNAs are very stable and no effect of the interferons on stability was detected. Nor was any direct evidence obtained for preferential processing of the 9-27 mRNA. Nevertheless, differentials between the transcription and accumulation of mature mRNAs, particularly for 6-16 mRNA in response to gamma interferon, suggest that post-transcriptional control(s) must additionally operate. The 9-27 5' promoter/enhancer is much less active than that from 6-16 when placed 5' of a marker gene, despite the similar response of the two genes to alpha interferon.
Blotting, Northern↗
Isolation and characterization of a new mutant human cell line unresponsive to alpha and beta interferons.
Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlled by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-alpha, we isolated 11.1, a recessive mutant that does not respond to IFN-alpha. We now describe U2, a second recessive mutant, selected similarly, that complements 11.1. U2 had no response to IFN-alpha or IFN-beta, and its response to IFN-gamma was partially defective. Although many genes did respond to IFN-gamma in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN-gamma was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-alpha (E and M) or IFN-gamma (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN-gamma but not IFN-alpha. Band shift complementation assays revealed that untreated and IFN-gamma-treated U2 cells lack the functional E gamma subunit of transcription factor E and that IFN-alpha-treated U2 cells do contain the functional E alpha subunit.
Aminacrine↗
Distinctive chromosomal structures are formed very early in the amplification of CAD genes in Syrian hamster cells.
As visualized by in situ hybridization with fluorescence detection, newly amplified CAD genes in 10(5) cell colonies are contained in multiple copies of very large regions of DNA, each tens of megabases long. The extra DNA is usually linked to the short arm of chromosome B9, which retains CAD at its normal site. The widely spaced genes are often interspersed with new G-negative regions. Individual cells within a clone have highly variable numbers of CAD genes (range 2-15). When resistant clones are examined later, at the 10(15) cell stage, the amplified genes are usually found in much more condensed structures. We propose that, in the initial event of CAD gene amplification, much of the short arm is transferred from one B9 chromosome to another. In subsequent cell cycles this initial duplication expands rapidly through unequal but homologous sister chromatid exchanges. Relatively rare secondary events lead to more condensed structures.
Animals↗
Transcription factors induced by interferons alpha and gamma.
Factors induced by interferons (IFNs) bind to the IFN-stimulated response elements (ISREs) of many genes. In human cells treated with type I (alpha, beta) IFN, factor E is induced in about 1 min and factor M after about 1 hr. Factor G is induced after about 1 hr in cells treated with type II (gamma) IFN. G and M have very similar positions in bandshift assays, sensitivities to cycloheximide, footprints on an ISRE and relative affinities for different ISREs. Four different patterns of expression were observed in different cell lines: E,M and G strongly induced; M and G strongly but E weakly; only E and M induced; only E induced. Transcription in response to IFN alpha is initiated by E and probably maintained by M since, in fibroblasts, M is present maximally when transcription is most active and declines together with transcription. In Bristol-8 cells, where induction of M is not detected, transcription is still induced by IFN alpha and still declines in its continued presence, suggesting that M is not essential for either process. A variant ISRE with two G-to-C mutations binds E especially weakly but M and G strongly. The mutations don't change the response of a reporter gene to IFN gamma but do abolish its response to IFN alpha, suggesting that the binding of M is not sufficient for the latter. G probably acts positively, since its appearance correlates well with induction of transcription by IFN gamma. A 39-bp ISRE from the 9-27 gene binds E much better than M or G. Conversely, a 39-bp ISRE from the 6-16 gene binds M and G much better than E. Different patterns of expression of E, M, and G and different affinities of these factors for alternative ISREs may play a part in modulating the relative responses of genes to type I and type II IFNs in vivo.
Base Sequence↗
High frequency of double drug resistance in the B16 melanoma cell line.
Methotrexate (MTX) and N-(phosphonacetyl)-L-aspartate (PALA) are two agents to which cellular resistance can be conferred by gene amplification, but they do not generally show cross resistance. However, combined treatment with these two agents produced drug resistant cells in the B16 melanoma cell line at a much higher frequency than would be expected if resistance to the two agents was totally independent. An isolated doubly resistant clone, B16-F1 MP, showed a high frequency of resistance to pyrazofurin and ouabain, which are also agents to which resistance can be conferred by gene amplification. Thus MTX combined with PALA selected cells with an 'amplificator' phenotype (an increased ability to amplify parts of the genome). These B16-F1 MP cells had a decreased ability to form experimental lung metastases compared with the parent line but this difference was not found in baby hamster kidney cells with the amplificator phenotype. The mechanism underlying drug resistance may need to be considered when designing combination chemotherapy.
Amides↗
DNA amplification is rare in normal human cells.
Three types of normal human cells were selected in tissue culture with three drugs without observing a single amplification event from a total of 5 x 10(8) cells. No drug-resistant colonies were observed when normal foreskin keratinocytes were selected with N-(phosphonacetyl)-L-aspartate or with hydroxyurea or when normal mammary epithelial cells were selected with methotrexate. Some slightly resistant colonies with limited potential for growth were obtained when normal diploid fibroblast cells derived from fetal lung were selected with methotrexate or hydroxyurea but careful copy-number analysis of the dihydrofolate reductase and ribonucleotide reductase genes revealed no evidence of amplification. The rarity of DNA amplification in normal human cells contrasts strongly with the situation in tumors and in established cell lines, where amplification of oncogenes and of genes mediating drug resistance is frequent. The results suggest that tumors and cell lines have acquired the abnormal ability to amplify DNA with high frequency.
Breast↗