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PubMed1995

The chemistry of single-stranded 4'-DNA radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage.

BACKGROUND: Deoxyribosylnucleotide radicals with a radical center at the 4'-position are important intermediates in radical-induced DNA strand cleavage. In the presence of O2, these DNA radicals yield cleavage products that are partly oxidized. In the past, the postulated peroxide intermediates could not be detected directly because they were unstable under the conditions of either radical generation, the work-up procedure, or the analytical techniques used. We set out to generate and analyze these crucial intermediates in radical-induced DNA strand cleavage under mild conditions. RESULTS: Photolysis experiments with modified single-stranded oligonucleotides generated 4'-DNA radicals that were trapped by O2. Using MALDI-MS, DNA peroxides could be detected directly. Depending upon the precursor, these peroxides are formed either before or after the cleavage of the single-stranded DNA radical. Reactions in the presence of 18O2 and/or H218O as well as subsequent transformations to the oxidized cleavage products confirmed the structure of the DNA peroxides. CONCLUSIONS: Our technique of selective DNA radical generation under mild conditions makes it possible to detect labile reaction products of single-stranded DNA radicals and to gain further insight into their cleavage reactions. In cases where a radical pair is formed, the shielding effect protects the DNA radical from external attack so that cleavage of the single strand competes successfully with trapping by O2. This shielding effect might be of general importance if the DNA radicals are generated by reagents that bind to the DNA.

Aerobiosis↗

PubMed1997

Extension of the replicative life span of human diploid fibroblasts by inhibition of the p33ING1 candidate tumor suppressor.

Previous studies suggest that tumor suppressors may play significant roles in blocking the growth of cells during cellular senescence. We therefore studied the potential involvement of a novel growth inhibitor and candidate tumor suppressor gene called ING1, which we have cloned recently (I. Garkavtsev, A. Kazarov, A. Gudkov, and K. Riabowol, Nat. Genet. 14:415-420, 1996), in the process of cellular senescence. Our results show that the RNA and protein levels of ING1 were 8- to 10-fold higher in senescent cells than in young, proliferation-competent human diploid fibroblasts. Expression of the nuclear p33ING1 protein was regulated during the cell cycle, reaching maximal levels during DNA synthesis. Chronic expression of antisense ING1 RNA reproducibly resulted in extension of the proliferative life span of normal human fibroblasts by approximately seven population doublings.

Antigens, Polyomavirus Transforming↗

PubMed2003

Distinct changes in gene expression induced by A-Myb, B-Myb and c-Myb proteins.

The c-Myb, A-Myb and B-Myb transcription factors have nearly identical DNA-binding domains, activate the same reporter gene constructs in animal cells, but have different biological roles. The Myb proteins are often coexpressed in the same cells, raising questions about whether they activate similar or distinct gene expression profiles, and whether they cooperate or compete in regulating the same promoters. Here, recombinant adenoviruses were used to express each protein in human mammary cells, and then microarray assays were used to assess global changes in gene expression. Each Myb protein induced a unique and specific set of changes, displaying activities far more complex than revealed by standard reporter gene assays. These results have important implications for the roles of various Myb proteins in normal and transformed human cells, for regulatory pathways that might modify their activities and for the importance of acquired mutations that may qualitatively alter their functions in tumors.

Adenoviridae↗

PubMed2001

Repression of dpp targets by binding of brinker to mad sites.

Signaling by decapentaplegic (Dpp), a Drosophila member of the transforming growth factor (TGF) beta superfamily of growth factors, has recently been shown to activate targets such as vestigial (vg) indirectly through negative regulation of brinker (brk). Here we show that the Brk protein functions as a repressor by binding to Dpp response elements. The Brk DNA binding activity was localized to an amino-terminal region containing a putative homeodomain. Brk bound to a Dpp response element of the Ultrabithorax (Ubx) midgut enhancer at a sequence that overlaps a binding site for the Smad protein, Mothers Against Dpp (Mad). Furthermore, Brk was able to compete with Mad for occupancy of this binding site. This recognition of overlapping binding sites provides a potential explanation for why the G/C-rich Mad binding site consensus differs the Smad3/Smad4 binding site consensus. We also found that the Dpp response element from Ubx was more sensitive than the vg quadrant enhancer to repression by Brk. This difference correlates with short-range activation of Ubx by Dpp in the visceral mesoderm, whereas vg exhibits a long-range response to Dpp in the wing imaginal disc, indicating that Brk binding sites may play a critical role in limiting thresholds for activation by Dpp. Finally, we provide evidence that Brk is capable of functioning as an active repressor. Thus, whereas Brk and Mad compete for regulation of Ubx and vg, Brk may regulate other Dpp targets without direct involvement of Mad.

Animals↗

PubMed1997

Overexpression of ZBP-89, a zinc finger DNA binding protein, in gastric cancer.

ZBP-89 is a Krüppel-type zinc finger protein that binds to the gastrin EGF response element (gERE). Sp1 binds to the same DNA element and transactivates gastrin promoter activity, whereas ZBP-89 competes for Sp1 binding and prevents EGF induction. Both transcription factors mediate growth factor signals originating from the EGF receptor and thus were studied in normal and neoplastic tissues or cell lines. When compared to normal tissue from the same patient, ZBP-89 protein expression was increased in neoplastic tissue from the stomach antrum and in malignant cell lines. RT-PCR analysis of ZBP-89 mRNA correlated with protein overexpression. Immunocytochemical studies confirmed that ZBP-89 expression is elevated in neoplastic tissue and chronic gastritis, whereas Sp1 expression was nearly unchanged. These results suggest that the transcription factor ZBP-89, like Sp1, may be a marker for neoplastic transformation in some gastric cancers.

Cells, Cultured↗

PubMed2004

Identification of a 98-kb DNA segment containing the rice Eui gene controlling uppermost internode elongation, and construction of a TAC transgene sublibrary.

The recessive 'tall rice' phenotype associated with the mutation eui (elongated upper-most internode) is an important agronomic trait that has been introduced into hybrid rice to eliminate panicle enclosure in all types of male-sterile lines and produce good-quality seeds in high yield and at low cost. Based on our previous Eui mapping data, we conducted fine-structure mapping and positional cloning of the gene using an F2 population comprising more than 5000 individuals derived from a cross of the near-isogenic lines 307T (eui/eui) with the recurrent parent Zhenshan 97 (Eui/Eui). In total 45 CAPS (cleaved amplified polymorphic sequences) markers located within an interval of 14.5 cM were analyzed in the subpopulation of 1298 homozygous recessive plants. The resulting high-resolution map defined a 98-kb interval containing the Eui locus flanked by the markers M0387 and M01, and three markers were found to co-segregate with Eui. In order to facilitate the identification of the Eui gene, we used a transformation-competent artificial chromosome (TAC) vector to construct a set of contiguous TAC clones from the Nipponbare BACs (obtained from the Clemson University Genome Institute; CUGI) spanning this region. These clones can be used to streamline complementation testing. The markers tightly linked to the Eui locus can also be used in breeding male-sterile lines with the elongated uppermost internode.

Base Sequence↗

PubMed1996

HIV-1 tat induces the expression of a new hematopoietic cell-specific transcription factor and downregulates MIP-1 alpha gene expression in activated T-cells.

MIP-1 alpha is a secreted chemokine which can inhibit hematopoietic stem cells and modulate inflammatory responses. It is also an inhibitor of HIV replication in CD8+ T-cells. The expression of MIP-1 alpha is induced during cellular activation of CD4+ T-cells and monocytes. It is also expressed in transformed B-cells. We have previously identified a new transcription factor family (the MNP family) whose expression is crucial for the induction of MIP-1 alpha transcription during cellular activation and in transformed B cells. Monocytes and transformed B-cells normally express MNP-1 strongly and MNP-2 weakly, while T-cells strongly express only MNP-2. Recently, we reported that HIV-1 tat downregulates MIP-1 alpha expression in Jurkat T-cells. In this report we show induction of MNP-1 in Jurkat T-cells expressing HIV-1 tat. Expression of neither HTLV-1 tax in Jurkat T-cells nor EBV in B-cells had any effect on MNP-1 or MNP-2 expression, showing that the effect is specific for HIV-1 tat. We propose that HIV-1 tat may inhibit MIP-1 alpha expression by inducing MNP-1 expression in T-cells, probably by either competing with MNP-2 for binding to the MIP-1 alpha promoter or by sequestering it into inactive forms.

B-Lymphocytes↗

PubMed1998

Cooperative interaction of autocrine and paracrine mitogens for airway epithelial cells.

Mitogens of the EGF family may play an important role in regulating the proliferation of airway epithelial cells (AEC). We examined the production of autocrine mitogenic activity by mouse AEC cultured from explants of tracheal tissue. DNA synthesis by growth-arrested AEC was stimulated by conditioned media from cells maintained in serum-free culture without exogenous growth factors. The mitogenic activity was blocked by a specific inhibitor of the EGF receptor tyrosine kinase. Furthermore, conditioned media from AEC contained molecular species that could compete with radiolabeled EGF in a receptor binding assay. However, mitogenic activity was not blocked by neutralizing antibodies to EGF or to transforming growth factor-alpha, but was partly inhibited by co-incubation with heparin, suggesting that it might be due to a heparin-binding member of the EGF family. The activity was potentiated by co-incubation with IGF-1, analogous to the potentiation by IGF-1 of the mitogenic activity of EGF for AEC. Moreover, the autocrine mitogen produced by AEC exhibited cooperative interaction with the mitogenic activity in conditioned media from growth factor-deprived mouse lung fibroblasts, consistent with the hypothesis that interactions with mesenchymal cells could influence the proliferation of AEC in vivo.

Animals↗

PubMed1985

HTLV x-gene product: requirement for the env methionine initiation codon.

The human T-cell leukemia viruses (HTLV) are replication-competent retroviruses whose genomes contain gag, pol, and env genes as well as a fourth gene, termed x, which is believed to be the transforming gene of HTLV. The product of the x gene is now shown to be encoded by a 2.1-kilobase messenger RNA derived by splicing of at least two introns. By means of S1 nuclease mapping of this RNA and nucleic acid sequence analysis of a complementary DNA clone, the complete primary structure of the x-gene product has been determined. It is encoded by sequences containing the env initiation codon and one nucleotide of the next codon spliced to the major open reading frame of the HTLV-I and HTLV-II x gene.

Amino Acid Sequence↗

PubMed1985

Mapping of transforming growth factor alpha gene on human chromosome 2 close to the breakpoint of the Burkitt's lymphoma t(2;8) variant translocation.

Transforming growth factors (TGFs) are defined as biologically active polypeptides which reversibly confer the transformed phenotype onto untransformed cultured cells. TGF-alpha shows sequence homology with epidermal growth factor and competes with epidermal growth factor for binding to the epidermal growth factor receptor, stimulating the phosphorylation of the receptor. TGF-alpha is secreted by many transformed cells and may be involved in embryonic development. A cloned human TGF-alpha gene was used to map the locus for the TGF-alpha precursor to the short arm of human chromosome 2, region 2p11----2p13, by Southern blotting techniques with DNA prepared from rodent X human somatic cell hybrids. These hybrids contained different subsets of human chromosomes and included a set with a translocation between human chromosomes 1 and 2 [t(1;2) (q32;q13)]. In situ hybridization of the TGF-alpha probe to normal human metaphase spreads confirmed these data and localized TGF-alpha more precisely to bands 2p11----2p13. Breakpoints in the variant Burkitt lymphoma translocation t(2;8) occur within these bands. Such a t(2;8) translocation could place TGF-alpha next to c-myc in band 8q24. The possibility is raised that TGF-alpha might contribute to tumor progression in these cases of Burkitt's lymphoma.

Burkitt Lymphoma↗

PubMed1975

Nucleolytic degradation of homologous and heterologous deoxyribonucleic acid molecules at the surface of competent pneumococci.

Competent pneumococci can catalyze the rapid and quantitative degradation of extracellular deocyribonucleic acid (DNA) molecules through the activity of surface-located nucleases (endo- and, possibly, exonucleases as well). Both homologous and heterologous DNAs are degraded by a mechanism that seems to involve a cyclic process: (i) attachment of DNA to the cell surface followed by (ii) nucleolytic attack, and (iii) release to the medium. Processes (ii) and (iii) are both inhibited by ethylenediaminetetraacetate. Whereas surface nuclease activity is specific for competent cells, the bulk of this activity is not coupled to irreversible DNA uptake (deoxyribonuclease-resistant binding). Pneumococcal DNA treated with ultraviolet irradiation or nitrous acid (cross-linking?) is selectively impaired in the ability to irreversibly bind to competent cells, whereas reversible binding is normal.

Adenoviridae↗

PubMed2004

Interconnection of competence, stress and CiaR regulons in Streptococcus pneumoniae: competence triggers stationary phase autolysis of ciaR mutant cells.

Of the 13 two-component signal transduction systems (TCS) identified in Streptococcus pneumoniae, two, ComDE and CiaRH, are known to affect competence for natural genetic transformation. ComD and ComE act together with the comC-encoded competence-stimulating peptide (CSP) and with ComAB, the CSP-dedicated exporter, to co-ordinate activation of genes required for differentiation to competence. Several lines of evidence suggest that the CiaRH TCS and competence regulation are interconnected, including the observation that inactivation of the CiaR response regulator derepresses competence. However, the nature of the interconnection remains poorly understood. Interpretation of previous transcriptome analyses of ciaR mutants was complicated by competence derepression in the mutants. To circumvent this problem, we have used microarray analysis to investigate the transition from non-competence to competence in a comC-null wild-type strain and its ciaR derivative after the addition of CSP. This study increased the number of known CSP-induced genes from approximately 47 to 105 and revealed approximately 42 genes with reduced expression in competent cells. Induction of the CiaR regulon, as well as the entire HrcA and part of the CtsR stress response regulons, was observed in wild-type competent cells. Enhanced induction of stress response genes was detected in ciaR competent cells. In line with these observations, CSP was demonstrated to trigger growth arrest and stationary phase autolysis in ciaR cells. Taken together, these data strongly suggest that differentiation to competence imposes a temporary stress on cells, and that the CiaRH TCS is required for the cells to exit normally from the competent state.

Bacterial Proteins↗

PubMed1964

CORRELATION BETWEEN GENETIC TRANSFORMABILITY AND NON-PHOTOREACTIVABILITY IN BACILLUS SUBTILIS.

Kelner, Albert (Brandeis University, Waltham, Mass.). Correlation between genetic transformability and nonphotoreactivability in Bacillus subtilis. J. Bacteriol. 87:1295-1303. 1964.-Photoreactivation after ultraviolet irradiation was studied in the transformable Bacillus subtilis SB-1. Moderate photoreactivability (maximal increase in survival due to photoreactivating light, five- to tenfold) was found in (i) noncompetent vegatative cells produced in Brain Heart Infusion broth, and in (ii) the total viable cells of a competent culture grown in special competency-producing medium and tested either just before or after transformation with deoxyribonucleic acid. However, no, or only trace, photoreactivation (maximal increase in survival, 1.5- to 2-fold) was found in that fraction of a competent culture actually able to be transformed. This nonphotoreactivability was a temporary property of transformable cells, since clones derived from them were normally photoreactivable. Nonphotoreactivability is not explained by injury to transformants caused by photoreactivating light, although transformants are killed by very large doses of light. The findings in B. subtilis strengthen the idea that transformability is generally correlated with nonphotoreactivability.

Bacillus subtilis↗

PubMed1995

Direct transfer of plasmid DNA between Streptomyces spp. and E. coli by electroduction.

We describe a simple and reliable method which allows the direct transfer of shuttle plasmids between Streptomyces spp. and E. coli. The method is based on the fact that plasmid DNA molecules can be released or taken up from cells under conditions of electroporation. When a suspension of a plasmid-containing Streptomyces spp. is mixed with electroporation-competent E. coli and submitted to an electric pulse, plasmid DNA transfer to the E. coli recipient takes place. Two different Streptomyces spp. (S. lividans TK23, or TK24 and S. rimosus 554w) were effective donors, and the method was successfully employed to transfer four different bifunctional vectors (pPM801, pFD666, pRL270X and pZG5) varying in size from 5.2-14.4 kb, to E. coli. This provides a convenient method for the analysis of Streptomycete transformants.

DNA, Bacterial↗

PubMed1970

Uptake of synthetic polynucleotides by competent cells of Bacillus subtilis.

A survey was made of the capacity of competent cells of Bacillus subtilis to take up synthetic polynucleotides. Polydeoxyribonucleotides but not polyribonucleotides are taken up by the cells. Both types of polynucleotide failed to compete with transforming deoxyribonucleic acid in the transformation assay whereas both stimulated amino acid incorporation. The latter phenomenon appears to be nonspecific as there is no correlation between the codon composition of the polynucleotides employed and the amino acids whose incorporation was stimulated.

Amino Acids↗

PubMed1975

[Suppression of the conjugational transfer of derepressed R factors by the dye, metachrome yellow].

Metachrome yellow widely used for preparation of nutrient media was tested with respect to its effect on conjugation of R-transfer. The standardized kinetic system described by the authors earlier was used. It was shown that metachrome yellow had a pronounced inhibitory effect on conjugation transfer of depressive R-factors RI drd and R 64 drd. The effect was markedly pronounced even in the presence of 100 gamma/ml of metachrome yellow, the concentration which was practically not bacteriostatic as it was shown in the analysis of the microbial growth curve in the presence of 100, 200 and 400 gamma/ml of the dye. The inhibition coefficients for 100 gamma/ml of metachrome yellow in the experiments with the donor of E. coli J 53 (R1 drd) were 11.8 and 12.1 times at 1-hour cultivation and t leads to infinity respectively, while in the experiments with the donor of E. coli J53 (R 64 drd) the respective values were 9.1 and 11.1. It was found that metachrome yellow induced no elimination of R-factors at low concentrations, such as 100 and 200 gamma/ml, which was indicative of the fact that the inhibitory effect on R-transfer was not connected with elimination of R-factors. It was also shown that preliminary cultivation of the donor in the presence of 100 and 400 gamma/ml of the dye did not induce any decrease in its donor competence which did not confirm the assumption of the inhibitory effect of metachrome yellow on synthesis of sex-pilei.

Coloring Agents↗

PubMed2001

Regulation of Smad degradation and activity by Smurf2, an E3 ubiquitin ligase.

Smad proteins are key intracellular signaling effectors for the transforming growth factor-beta superfamily of peptide growth factors. Following receptor-induced activation, Smads move into the nucleus to activate transcription of a select set of target genes. The activity of Smad proteins must be tightly regulated to exert the biological effects of different ligands in a timely manner. Here, we report the identification of Smurf2, a new member of the Hect family of E3 ubiquitin ligases. Smurf2 selectively interacts with receptor-regulated Smads and preferentially targets Smad1 for ubiquitination and proteasome-mediated degradation. At higher expression levels, Smurf2 also decreases the protein levels of Smad2, but not Smad3. In Xenopus embryos, ectopic Smurf2 expression specifically inhibits Smad1 responses and thereby affects embryonic patterning by bone morphogenetic protein signals. These findings suggest that Smurf2 may regulate the competence of a cell to respond to transforming growth factor-beta/bone morphogenetic protein signaling through a distinct degradation pathway that is similar to, yet independent of, Smurf1.

Acetylcysteine↗

PubMed1997

Characterization of downstream Ras signals that induce alternative protease-dependent invasive phenotypes.

Invasive and metastatic cells require protease expression for migration through the extracellular matrix. Metastatic NIH 3T3 fibroblasts transformed by different activated ras genes showed two different protease phenotypes, rasuPA+/CL- and rasCL+/uPA- (Zhang, J-Y., and Schultz, R. M. (1992) Cancer Research 52, 6682-6689). Phenotype rasuPA+/CL- is dependent on expression of the serine-type protease urokinase plasminogen activator (uPA) and the phenotype rasCL+/uPA- on the cystine-type protease cathepsin L (CL) for lung colonization in experimental metastasis. The existence of multiple invasive phenotypes on ras-isoform transformation implied the activation of alternative pathways downstream from Ras. We now show that c-Raf-1, extracellular signal-regulated protein kinase (ERK)-1, and ERK-2 are hyperphosphorylated, and the ERK activity is high in both the uPA- and CL-dependent ras-transformed invasive phenotypes. Levels of c-Jun and c-Jun NH2-terminal kinase (JNK) activity are also high in the uPA-dependent phenotype, but they are almost undetectable in the CL-dependent phenotype. The uPA Ras-response element is a PEA3/URTF element, and mobility shift assays show a strong PEA3/URTF protein band in the uPA-dependent phenotype. This band is competed by a consensus AP-1 DNA sequence and by antibodies to PEA3 and c-Jun. Thus, the uPA-invasive phenotype appears to require the activation of Ets/PEA3 and c-Jun transcription factors activated by the ERK and JNK pathways, while the CL-invasive phenotype appears to require ERK activity with suppression of JNK and c-Jun activities. These postulates are supported by the introduction of a dominant negative c-Jun, TAM67, into cells of phenotype rasuPA+/CL-, which down-regulated the high uPA mRNA levels characteristic of this phenotype to basal levels and up-regulated basal levels of CL mRNA to levels similar to those observed in cells of phenotype rasCL+/uPA-. We conclude that the JNK pathway acts as a switch between two distinct protease phenotypes that are redundant in their abilities to grow tumors and metastasize.

3T3 Cells↗