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At least 883 records · Page 49Linked to original sources
PubMed1992

Transcriptional regulation of the human IgE receptor (Fc epsilon RII/CD23) by EBV. Identification of EBV-responsive regulatory elements in intron 1.

EBV infection of B cells induces the B cell activation Ag, CD23 (Fc epsilon RII). CD23 remains constitutively expressed at high levels in all EBV-immortalized B cells and likely plays an important role in the initiation and maintenance of immortalization by EBV. By utilizing an EBV-negative Burkitt's lymphoma line (BJAB) and EBV-positive sublines derived from it by in vitro infection, we have examined the molecular mechanisms involved in the regulation of CD23 by EBV. By nuclear runoff analysis, we have found that induction of CD23 is mediated by transcriptional activation that occurs in the presence of the transformation-competent B958 virus but not in the presence of the nontransforming P3HR-1 strain of EBV. To identify EBV-responsive transcriptional regulatory elements of CD23, we have performed reporter gene assays using plasmids containing fragments of the CD23 gene derived from its 5' terminus and adjacent flanking region transfected into EBV-positive and -negative BJAB lines. We have identified a 534-bp fragment of the gene which enhances transcription from a heterologous promoter (SV40) and reporter gene (chloramphenicol acetyltransferase) only in the presence of transformation-competent strains of EBV. Deletion of 144 bp of intron 1 from the 3' end of this fragment results in loss of EBV-responsive enhancer activity. The finding of an EBV-responsive enhancer element of CD23 is supported by mobility shift assays that demonstrated the formation of specific DNA-protein complexes between nuclear protein from transforming EBV-positive cells and the 144-bp intron sequence. These studies suggest that the transcriptional activation of CD23 by transforming strains of EBV involves regulatory elements that are located within the first intron of the gene.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1989

High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier.

A method, using LiAc to yield competent cells, is described that increased the efficiency of genetic transformation of intact cells of Saccharomyces cerevisiae to more than 1 X 10(5) transformants per microgram of vector DNA and to 1.5% transformants per viable cell. The use of single stranded, or heat denaturated double stranded, nucleic acids as carrier resulted in about a 100 fold higher frequency of transformation with plasmids containing the 2 microns origin of replication. Single stranded DNA seems to be responsible for the effect since M13 single stranded DNA, as well as RNA, was effective. Boiled carrier DNA did not yield any increased transformation efficiency using spheroplast formation to induce DNA uptake, indicating a difference in the mechanism of transformation with the two methods.

DNA, Fungal↗
PubMed1998

Stable electrotransformation of symbiont candidate diazotrophic bacterium with plasmids carrying selectable and screenable marker genes.

Nitrogen-fixing symbioses had been established between the originally asymbiotic soil bacterium Azotobacter vinelandii CCM289 and different lower and higher plant species. Better characterization and further development of such artificial systems require a reliable genetic transformation method for the introduction of marker genes into symbiont candidates. The performance of electroporation was evaluated using pJB3 (4.8 kb), pBI121 (12.8 kb) and pFAJ31.2 (24 kb) plasmid DNAs containing selectable (Ap, Km, Tc) and screenable (gusA, lacZ) marker genes. The adapted methods for the preparation of transformation-competent azotobacters and their electroporation (18 kV/cm electric field strength, 5 ms time constant, 0 degree C) provided up to 6.8 x 10(5) transformants per microgram plasmid DNA, which is about 10(3) times the transformation efficiency achieved in control experiments. No electrotransformants were obtained with the 24-kb pFAJ31.2. The size of plasmid DNA did not significantly affect the efficiency of transformation. Transformants were able to grow at antibiotic concentrations that were 100-200 times greater than the lowest amounts that completely inhibited the growth of wild-type bacteria. A constitutive expression of gusA gene was observed in transformants with the CaMV 35S promoter-gusA fusion containing pBI121, while lacZ expression was not detected under the control of the lac promoter in pJB3 transformants. Electroporated plasmids were reisolated from transformants in their original form, while non-transformed bacteria did not contain indigenous plasmids. PCR amplification and Southern DNA blot hybridization showed the integration of plasmid DNA into the host genome as well. Transformants retained their nitrogen-fixing ability and had normal morphological and growth characteristics. Experimental findings proved the stable maintenance of plasmid DNA in azotobacters, making possible the routine transformation and detection of these symbiont candidates.

Azotobacter vinelandii↗
PubMed1998

Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae.

Transcriptional activation of the recA gene of Streptococcus pneumoniae was previously shown to occur at competence. A 5.7 kb recA-specific transcript that contained at least two additional genes, cinA and dinF, was identified. We now report the complete characterization of the recA operon and investigation of the role of the competence-specific induction of recA. The 5.7 kb competence-specific recA transcript is shown to include lytA, which encodes the pneumococcal autolysin, a protein previously shown to contribute to virulence of S. pneumoniae. Uncoupling (denoted Ind-) of recA and/or the downstream genes was achieved through the placement of transcription terminators within the operon, either upstream or downstream of recA. Prevention of the competence-specific induction of recA severely affected spontaneous transformation. Transformation efficiencies of recA+ (Ind-) and of wild-type cells were compared under various conditions and with different donor DNA. Chromosomal transformation was reduced 17-(chromosomal donor) to 45-fold (recombinant plasmid donor), depending on the donor DNA, and plasmid establishment was reduced 129-fold. Measurement of uptake of radioactively labelled donor DNA in transformed cells in parallel with scoring for transformants (chromosomal donor) revealed normal uptake, but a 21-fold reduction in recombination in a recA+ (Ind-) strain, indicating that the transformation defect was primarily in recombination. Strikingly enough, a much larger (460-fold) reduction in recombination was observed for the shortest homologous donor fragment used (878 nucleotides long). Possible interpretations of the observation that basal RecA appears unable to promote efficient recombination whatever the number and the length of donor fragments taken up are proposed. The role of recA induction is discussed in view of the potential contribution of transformation to genome plasticity in this pathogen.

Amidohydrolases↗
PubMed1965

PROPERTIES OF NEWLY INTRODUCED TRANSFORMING DEOXYRIBONUCLEIC ACID IN BACILLUS SUBTILIS.

Venema, G. (Hammersmith Hospital, London, England), R. H. Pritchard, and T. Venema-Schröder. Properties of newly introduced transforming deoxyribonucleic acid in Bacillus subtilis. J. Bacteriol. 90:343-346. 1965.-In these experiments, deoxyribonucleic acid (DNA) with donor markers, re-extracted shortly after uptake by competent Bacillus subtilis cells, had abnormal physical properties as compared with DNA with resident markers. It was less sensitive to thermal denaturation, and its capacity to renaturation was reduced. DNA showing joint activity of donor and recipient markers (recombinant-type activity) had similar abnormal properties, revealing that, despite the appearance of recombinant-type activity, integration was not complete.

Bacillus subtilis↗
PubMed2002

Prolonged arrest of mammalian cells at the G1/S boundary results in permanent S phase stasis.

Mammalian cells in culture normally enter a state of quiescence during G1 following suppression of cell cycle progression by senescence, contact inhibition or terminal differentiation signals. We find that mammalian fibroblasts enter cell cycle stasis at the onset of S phase upon release from prolonged arrest with the inhibitors of DNA replication, hydroxyurea or aphidicolin. During arrest typical S phase markers remain present, and G0/G1 inhibitory signals such as p21(WAF1) and p27 are absent. Cell cycle stasis occurs in T-antigen transformed cells, indicating that p53 and pRB inhibitory circuits are not involved. While no DNA replication is evident in arrested cells, nuclei isolated from these cells retain measurable competence for in vitro replication. MCM proteins are required to license replication origins, and are put in place in nuclei in G1 and excluded from chromatin by the end of replication to prevent rereplication of the genome. Strikingly, MCM proteins are strongly depleted from chromatin during prolonged S phase arrest, and their loss may underlie the observed cell cycle arrest. S phase stasis may thus be a 'trap' in which cells otherwise competent for S phase have lost a key component required for replication and thus can neither go forward nor retreat to G1 status.

Animals↗
PubMed2005

Poly-(R)-3-hydroxybutyrate and the pioneering work of Rosetta Natoli Reusch.

In investigating genetic competence, Reusch and collaborators have found that the concentration of short chain poly-(R)-3-hydroxybutyrate (PHB) and polyphosphate (polyP) complexes increases with genetic transformability and that interrupting DNA uptake yields single-stranded donor DNA complexed with short chain PHB. This would be consistent with the organic polyphosphate, DNA, replacing the inorganic polyphosphate, polyP, in the PHB pore so allowing the DNA to be drawn into the cell. Reusch has gone on to show that PHB and polyphosphate, extracted from membranes or synthesized chemically, together form a voltage-activated calcium-selective channel. One may wonder whether the classical proteinaceous calcium channels have a short chain PHB/polyP core--and whether other ion channels have this core too. It is therefore significant that in Streptomyces lividans the potassium channel KcsA, which resembles that of eukaryotes, forms tetramers that contain polyP whilst both monomers and tetramers are covalently linked to short chain PHB. Pumps are the counterparts of channels. Reusch has also shown that a model pump, the calcium ATPase pump of human erythrocytes, contains both cPHB and polyP and has strongly implicated these polymers in its functioning. Again, one may wonder whether these polymers are essential constituents of other pumps. Reusch has gone on to show that a wide range of proteins are modified post-translationally by covalent addition of short chain PHB in both prokaryotes and eukaryotes including DNA-binding proteins such as histones. Finally, Reusch has extended the importance of short chain PHB to medicine by showing its likely involvement in atherogenic plaques and diabetes. And yet this opus has gone largely unnoticed.

3-Hydroxybutyric Acid↗
PubMed1993

Similar simian virus 40-induced immortalization frequency of fibroblasts and epithelial cells from human large airways.

Several previous studies have shown that when Simian virus 40 (SV40) genes were introduced into normal human fibroblasts, a very small proportion (< 2%) of the resulting transformed clones were immortalization-competent. In separate studies with normal human epithelial cells, SV40 genes were shown to induce a much higher proportion (30-100%) of immortalization-competent clones. In this study, a direct comparison was made of the immortalization frequencies of clones of normal epithelial cells and fibroblasts derived from the same individual. The results demonstrate that fibroblasts and epithelial cells can be immortalized at similarly high frequencies when transfected with an SV40 early region origin-minus expression plasmid, without the need for a selectable marker. We conclude that there is no intrinsic difference between the SV40 early region-induced immortalization potentials of human fibroblasts and epithelial cells. Comparison of this study with other studies of the immortalization of human fibroblasts suggests that removal of SV40 late region genes from the transfected plasmid is important for obtaining a high proportion of immortalization-competent clones.

Antigens, Polyomavirus Transforming↗
PubMed1990

Alpha-transforming growth factorlike activities and bifunctional regulators of cell growth in human malignant neoplasms.

Multiple transforming growth factors (TGFs) capable of conferring the neoplastic phenotype on NRK-49F cells without the addition of any other exogenous growth factor in the soft agar assay, were purified from two human solid malignant neoplasms: a squamous lung carcinoma and a pectoral rhabdomyosarcoma. In both tumors, low-molecular-weight transforming activities (4000-6000) that were not potentiated by epidermal growth factor (EGF), competed for binding to the EGF receptor, possessed mitogenic activity on NRK fibroblasts arrested in serum-deprived medium, and did not show inhibitory effects on DNA synthesis induced by EGF and insulin in NRK cells. Other TGFs with molecular weights 9000 to 48,000, were also found in the malignant tissues examined; these TGFs, were not potentiated by EGF, did not compete for binding to the EGF receptor, were not mitogenic for NRK cells, and acted as potent inhibitors of DNA synthesis induced by EGF and insulin in NRK cells. These results demonstrate that growth-promoting activities, and modulating agents that can act as either enhancers or inhibitors of cell proliferation, are present in neoplastic tissues of different embryologic origin and histologic type.

Carcinoma, Squamous Cell↗
PubMed1993

Interleukin 6 induces DNA binding activity of AP1 in M1 myeloblastic cells but not in a growth resistant cell derivative.

The effects that three different growth inhibitory cytokines exert on expression and function of members of the Jun family were studied in this work. M1 myeloblastic cells were chosen for this purpose because of their high growth sensitivity to interleukin 6 (IL-6), transforming growth factor beta 1 and alpha- and beta-interferons. It is reported here that IL-6 elevated the junB and c-jun mRNA levels and induced the formation of a novel DNA-protein complex with high sequence specificity to 12-O-tetradecanoylphorbol-13-acetate response element (TRE) oligonucleotides. This IL-6 induced TRE binding complex was abolished by anti-Jun specific antibodies and was efficiently competed by an oligonucleotide that comprises the mouse homologue of a previously described human c-myc negative DNA element. It persisted in cells for at least 48 h after IL-6 treatment and failed to be induced by alpha- and beta-interferons or by transforming growth factor beta 1, which affected differently the pattern of jun mRNA expression. To further explore regulatory and functional aspects of this induced TRE binding activity, an IL-6 resistant M1 clone was isolated and further analyzed. This clone carried a postreceptor deficiency that abrogated completely the growth inhibitory responses to IL-6 but did not interfere with the induction of two differentiation related cell surface markers. Interestingly, the IL-6 resistant clone had lost two molecular responses to IL-6, induction of TRE binding activity and suppression of the c-myc gene. The data correlate the IL-6 induced AP1 activity with the suppression of c-myc and growth inhibition.

Animals↗
PubMed1994

Adenovirus 12-mediated down-regulation of the major histocompatibility complex (MHC) class I promoter: identification of a negative regulatory element responsive to Ad12 E1A.

In highly oncogenic adenovirus (Ad) 12-transformed cells, major histocompatibility complex (MHC) class I gene expression is down-regulated by the products of the viral E1A oncogene at the level of initiation of transcription. However, class I gene expression is unaltered or elevated in non-oncogenic Ad2- or Ad5-transformed cells. These changes in class I expression may permit Ad12-transformed cells to escape host immune surveillance and elicit tumour formation. Here we show that the 2kb of 5' flanking region of the mouse H-2Kb class I gene is sufficient to mediate down-regulation of transcription driven from homologous or heterologous (HSV thymidine kinase) basal promoter elements in cells expressing Ad12 E1A, but not in Ad2 E1A-expressing cells. Deletion analysis of the 2kb region showed that sequences from -1.18 to -1.44kb (relative to the cap site) were a target for Ad12 E1A-mediated transcriptional down-regulation. Deletion of this entire region from the 2kb flanking sequence of the H-2Kb gene abolished Ad12 E1A-mediated down-regulation of transcription. Computer analysis of the -1.18 to -1.44kb sequence identified two 6/7bp matches with the AP-1 transcription factor consensus sequence and two matches with the pig MHC class I PD1 repressor element. Gel retardation analysis using overlapping DNA fragments derived from the -1.18 to -1.44kb sequence revealed several DNA:protein complexes formed using nuclear extract derived from Ad12-, but not from Ad2- or Ad5-transformed cells. Some of these DNA:protein complexes were also present, but at lower levels, in nuclear extracts from untransformed rat cells suggesting the possible involvement of cellular factors in the mechanism of down-regulation mediated by Ad12 E1A. A binding site for the AP-1 factor failed to compete for protein binding to fragments within the -1.18 to -1.44 sequence, while the PD1 site competed for binding only in the -1.15 to -1.23 region. These results indicate that novel factors (as well as a previously identified class I repressor, PD1) may be involved in Ad12 E1A-mediated down-regulation of MHC class I transcription.

Adenovirus E1A Proteins↗
PubMed1971

Early intermediate state of transforming deoxyribonucleic acid during uptake by Bacillus subtilis.

Examination of the effects of ethylenediaminetetraacetic acid (EDTA) on uptake of transforming deoxyribonucleic acid (DNA) by Bacillus subtilis allowed definition of a new intermediate state of transforming DNA during uptake. Markers in this state, termed "EDTA-resistant," are bound to the cell, are sensitive to inactivation by deoxyribonuclease, but may become deoxyribonuclease-resistant despite the addition of excess EDTA. Markers become EDTA-resistant quadratically with time after the addition of DNA to a competent culture, but linearly after exposure of competent cells to a brief pulse of DNA. An attractive model consistent with these findings is that EDTA blocks only the initiation of entry of a molecule of DNA.

Bacillus subtilis↗
PubMed1999

Long range cooperative interactions regulate the initiation of replication in the Tetrahymena thermophila rDNA minichromosome.

The Tetrahymena thermophila rDNA exists as a 21 kb palindromic minichromosome with two initiation sites for replication in each half palindrome. These sites localize to the imperfect, repeated 430 bp segments that include the nucleosome-free domains 1 and 2 (D1 and D2). To determine if the D1 and D2 segments act independently or in concert to control initiation, stable DNA transformation assays were performed. Single domain derivatives of the plasmid prD1 failed to support autonomous replication in Tetrahymena. Instead, such constructs propagated exclusively by integration into endogenous rDNA minichromosomes and displayed weak origin activity as detected by 2D gel electrophoresis. D1/D1 and D2/D2 derivatives also transformed Tetrahymena poorly, showing similar replication defects. Hence, the D1 and D2 segments are functionally non-redundant and cooperate rather than compete to control initiation. The observed replication defect was greatly reduced in a plasmid derivative that undergoes palindrome formation in Tetrahymena, suggesting that a compensatory mechanism overcomes this replication block. Finally, using a transient replication assay, we present evidence that phylogenetically-conserved type I elements directly regulate DNA replication. Taken together, our data support a model in which cooperative interactions between dispersed elements coordinately control the initiation of DNA replication.

Animals↗
PubMed1990

Production and response of a human prostatic cancer line to transforming growth factor-like molecules.

Serum-free media conditioned by the androgen insensitive human prostate cancer cell line DU145 showed immunological transforming growth factor-alpha (TGF alpha) activity, as well as competing activity in epidermal growth factor (EGF) radioreceptor assays (RRA). Furthermore, there were factors in the conditioned media which inhibited and stimulated DNA synthesis by DU145 cells in a dose-dependent fashion. Fractionation of the concentrated conditioned media by reverse-phase high performance liquid chromatography revealed several peaks containing EGF-like competitive activity only one of which demonstrated TGF alpha activity. However, none of the peaks corresponded to immunoreactive EGF. Measurement of EGF receptors on DU145 cells by competition and saturation analysis revealed high levels of receptors (mean +/- s.d. = 2.5 +/- 1 x 10(5) surface receptors per cell) which were of high affinity (Kd +/- s.d. = 1.0 +/- 0.5 nmol l-1). Although DU145 cells express high levels of EGF receptors, DNA synthesis was only minimally affected by exogenous EGF and TGF alpha.

Chromatography, High Pressure Liquid↗
PubMed1987

Site-specific gap-misrepair mutagenesis by O4-ethylthymine.

The mutagenicity of the DNA base O-alkylation adduct, O4-ethylthymine, specifically incorporated into the plasmid vector pUC8 at the unique SalI and HincII recognition sites, was studied in vivo. Escherichia coli, Micrococcus luteus and AMV DNA polymerases catalyze the incorporation of O4-ethylTMP against template adenine and guanine residues, resulting in DNA sequence alteration during subsequent replication in the host E. coli K-12 strain JM83. The greatest mutation frequency was observed with error-prone AMV DNA polymerase. High levels of cognate restriction endonuclease-resistant mutant plasmid isolates were obtained by gap replication repair in the presence of O4-ethylTTP. The yields of mutant isolates were dependent upon the relative concentration of the competing pyrimidine deoxynucleoside triphosphates, TTP and dCTP, in the misreplication reaction. Repair of incorporated O4-ethylTMP of plasmid DNA by in vitro treatment with specific alkyltransferase, prior to transformation in the host, effectively increases the mutagenic efficiency of the adduct. The results obtained are consistent with the high miscoding potential O4-ethylthymine observed in in vitro studies and its ability to base-pair with noncomplementary guanine residues in DNA.

Alkyl and Aryl Transferases↗
PubMed2003

Structural analysis of a Lotus japonicus genome. V. Sequence features and mapping of sixty-four TAC clones which cover the 6.4 mb regions of the genome.

We determined the nucleotide sequences of 64 TAC (transformation-competent artificial chromosome) clones selected from genomic libraries of Lotus japonicus accession Miyakojima MG-20 based on the sequence information of expressed sequence tags (ESTs), cDNAs, genes and DNA markers from L. japonicus and other legumes. The length of the DNA regions sequenced in this study was 6,370,255 bp, and the total length of the L. japonicus genome sequenced so far is 32,537,698 bp together with the nucleotide sequences of 256 TAC clones previously reported. Five hundred forty-eight potential protein-encoding genes with known or predicted functions, 127 gene segments and 224 pseudogenes were assigned to the newly sequenced regions by computer prediction and similarity searches against the sequences in protein and EST databases. Based on the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was genetically localized onto the linkage map of two accessions of L. japonicus, MG-20 and Gifu B-129. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

Base Sequence↗
PubMed1983

Genetic transformation of obligately chemolithotrophic thiobacilli.

Genetic transformation of Thiobacaillus thioparus auxotrophs to prototrophy was obtained at frequencies of up to 10(-2) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent strain of the same bacterium. The rate at which transformation occurred depended on recipient growth rate and could be drastically reduced by depriving otherwise competent cells of either nitrogen or exogenous energy substrate. Interspecies marker transfer was also shown among several obligately chemolithotrophic members of the genus.

Genetic Markers↗
PubMed2005

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.

Cell Line, Tumor↗