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PubMed1986

Distinct regions of the human cytomegalovirus genome are responsible for the immortalization and tumorigenicity of animal cells.

Dog embryo kidney cells were efficiently transformed by human cytomegalovirus (HCMV) particles or intact viral DNA. Negative results were obtained after transfection of the canine cells with recombinant plasmids carrying the HCMV Hind III-E subgenomic fragment or with limit Bgl II and Hind III digests of the viral genome. Immortalized dog cells with typical transformation properties appeared, however, after transfection with EcoR I fragments of the HCMV DNA. Distinct regions of the viral genome are probably responsible for the immortalization and the tumorigenicity of mammalian cells.

Cell Line↗

PubMed1984

Length of foreign DNA in chimeric plasmids determines the efficiency of its integration into the chromosome of the cyanobacterium Synechococcus R2.

The photosynthetic cyanobacterium Synechococcus R2 is efficiently transformed by DNA molecules that contain antibiotic genes from Escherichia coli linked to a segment of Synechococcus R2 chromosomal DNA. Antibiotic-resistant transformants result from integration of donor DNA into the cyanobacterial chromosome by homologous recombination. Foreign DNA interrupting the cyanobacterial sequence in the donor molecule integrates by replacement of homologous recipient DNA with donor DNA containing the foreign insert. Foreign DNA linked to the ends of the cyanobacterial fragment in a circular donor molecule integrates by a reciprocal cross-over between donor and recipient sequences. Using donor molecules that contain different lengths of foreign DNA in both of the above positions, we have determined that the probability of integration decreases by half for each increase of 2 to 3 kb in length of a foreign segment, regardless of its position in the donor molecule. The length of one of the two foreign segments has no effect on the integration of the other. Foreign DNA 20 kb in length is completely stable when it has integrated by the replacement mechanism. The ability to stably introduce large pieces of foreign DNA makes Synechococcus R2 an attractive organism in which to study and modify both native and heterologous genes involved in oxygenic photosynthesis.

Cyanobacteria↗

PubMed1985

Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences.

We describe the construction and properties of pEMBLY plasmids. They belong to a new family of yeast shuttle vectors which are derived from plasmid vector pEMBL9 and offer the following improvement: relatively small size; large number of cloning sites; screening for insert-containing plasmids on indicator plates; different combinations of genes which complement auxotrophic deficiencies and sequences that support DNA replication in Saccharomyces cerevisiae; and ability to isolate the plasmid DNA in single-stranded (ss) form. The yeast S. cerevisiae can be efficiently transformed by these plasmids in both the ss and double-stranded (ds) forms. Finally, the presence of the phage f1 intergenic region allows one to obtain the cloned sequences in the ss form upon infection with the wild-type ss phage [Dotto et al., Virology 114 (1981) 463-473].

Base Sequence↗

PubMed1986

Structure and activity of the translocated c-myc in mouse plasmacytoma XRPC-24.

In the mouse plasmacytoma XRPC-24 both c-mos and c-myc are rearranged. We cloned the rearranged c-myc and found that it was translocated to the immunoglobulin C alpha locus. The breakpoint is at the end of exon 1 in c-myc and approximately 0.5 kb upstream from exon 1 of C alpha. The cloned translocated c-myc linked to a strong transcriptional promoter can efficiently transform rat embryo fibroblasts when co-transfected with the activated Ha-ras. The transformed cells are tumorigenic in syngeneic rats.

Animals↗

PubMed1987

Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli.

An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.

Acetyltransferases↗

PubMed1988

New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites.

We describe the production of new alleles of the LEU2, URA3 and TRP1 genes of Saccharomyces cerevisiae by in vitro mutagenesis. Each new allele, which lacks restriction enzyme recognition sequences found in the pUC19 multicloning site, was used to construct a unique series of yeast-Escherichia coli shuttle vectors derived from the plasmid pUC19. For each gene a 2 mu vector (YEplac), an ARS1 CEN4 vector (YCplac) and an integrative vector (YIplac) was constructed. The features of these vectors include (i) small size; (ii) unique recognition site for each restriction enzyme found in the pUC19 multicloning site; (iii) screening for plasmids containing inserts by color assay; (iv) high plasmid yield; (v) efficient transformation of S. cerevisiae. These vectors should allow greater flexibility with regard to DNA restriction fragment manipulation and subcloning.

Alleles↗

PubMedDate not supplied

Characterization of a linear extrachromosomal DNA element (pBL1) isolated after interspecific mating between Streptomyces bambergiensis and S. lividans.

Streptomyces bambergiensis S712 harbours a giant linear plasmid PSB1 of 640 kb. After mating with the plasmidless S. lividans strain TK64, conjugants carrying a smaller extrachromosomal DNA element, pBL1, were identified. pBL1 is a 43-kb linear DNA molecule bound to a protein which protects it from attack by both 3'- and 5'-exonucleases. The absence of this protein drastically reduces the transforming efficiency of pBL1. pBL1 shares homology with linear plasmids and chromosomal DNA from S. bambergiensis strains.

Conjugation, Genetic↗

PubMed1993

Chlamydomonas phototaxis.

Chlamydomonas has long been a favourite organism for genetic and biochemical studies of flagellar motility and assembly, photosynthesis, and organelle genomes. With the recent development of procedures for the efficient transformation of its nuclear genome, Chlamydomonas has become accessible to a wide range of molecular genetic approaches, including gene tagging by insertional mutagenesis and cloning by complementation. The availability of these powerful techniques is stimulating interest in Chlamydomonas as a model system for research in areas where it previously has not been widely exploited. One such area that holds particular promise is phototransduction and the behavioural response to light.

Journal Article↗

PubMed1991

Biological consequences associated with DNA oxidation mediated by singlet oxygen.

Singlet oxygen is a major oxidative species that can be generated by numerous biological processes such as photosensitization. This oxidant can react with deoxyguanosine and with guanine in deoxyribonucleic acid (DNA) leading to the induction of at least four different reaction products such as 4,8-dihydro-4-hydroxy-8-oxodeoxyguanosine and 7,8-dihydro-8-oxodeoxyguanosine. The induction of true single-stranded breaks in the oxidated DNA is still a matter of controversy and is not yet clearly established. This paper focuses mainly on several biological consequences which can be associated with the induction of DNA lesions by singlet oxygen. Oxidated DNA loses its transformation efficiency probably because unrepaired lesions can partially inhibit DNA replication. Mutagenesis is one of the main effects induced by guanine oxidation products. Molecular analysis of mutated genes reveals that G to T transversions are the most frequent mutations; these are probably introduced in DNA by misincorporation of deoxyadenosine monophosphate (dAMP) opposite to the lesion. Efficient repair of these oxidated guanine residues can take place via specific glycosylase, endonuclease or the SOS network. However, the data concerning the toxicity of singlet oxygen for eukaryotic cells are not frequent enough in the literature to draw a clear picture of the effects of this activated species in several biologically revelant phenomena.

DNA Damage↗

PubMed2004

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode.

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.

Alcohol Oxidoreductases↗

PubMed2006

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.

Cell Line, Tumor↗

PubMed2004

Cells synchronized in S phase show increased rate of repair of UV damaged plasmids.

The capacity for nucleotide excision repair of cells synchronized in S phase and unsynchronized cells was compared by the host cell reactivation assay and the cell-free repair system. HeLa cells were transfected with in vitro damaged by UV irradiation pEGFP and the repair capacity was determined by the number of fluorescent cells. In the cell-free repair system, the repair capacity of protein extracts isolated from K562 cells was determined by measuring the transformation efficiency of UV irradiated pBlueScript incubated in the extracts. In both cases, the repair capacity of the cells synchronized in S phase cells was 30-50% higher than the repair capacity of unsynchronized cells.

Cell Cycle↗

PubMed2005

Over-expression of xylulokinase in a xylose-metabolising recombinant strain of Zymomonas mobilis.

The broad host range vector pBBR1MCS-2 has been evaluated as an expression vector for Zymomonas mobilis. The transformation efficiency of this vector was 2 x 10(3) CFU per mug of DNA in a recombinant strain of Z. mobilis ZM4/AcR containing the plasmid pZB5. Stable replication for this expression vector was demonstrated for 50 generations. This vector was used to study xylose metabolism in acetate resistant Z. mobilis ZM4/AcR (pZB5) by over-expression of xylulokinase (XK), as previous studies had suggested that XK could be the rate-limiting enzyme for such strains. Based on the above vector, a recombinant plasmid pJX1 harboring xylB (expressing XK) under control of a native Z. mobilis promotor Ppdc was constructed. When this plasmid was introduced into ZM4/AcR (pZB5) a 3-fold higher XK expression was found compared to the control strain. However, fermentation studies with ZM4/AcR (pZB5, pJX1) on xylose medium did not result in any increase in rate of growth or xylose metabolism, suggesting that XK expression was not rate-limiting for ZM4/AcR (pZB5) and related strains.

Base Sequence↗

PubMed2026

Multiplexed CRISPR/Cas9 mediated knockdown of BCH gene in potato enhances beta-carotene to combat vitamin A deficiency.

The inadequate amounts of provitamin A carotenoids in crops contribute to the widespread vitamin A deficiency, leading to malnutrition and blindness in humans. Suppression of the β-carotene hydroxylase (BCH) increases β-carotene levels. In the current study, we utilized the multiplexed CRISPR/Cas9 approach by designing three targets against the BCH gene in a local potato cultivar. Transformation efficiency was recorded as 15%, the successful integration of the CRISPR/Cas9-BCH multiplex construct in potatoes was confirmed through PCR. When analysed using TIDE software, Sanger sequencing revealed the highest indel efficacy of 92.1% in plant 7 and 26.6% in plant 1. qRT-PCR (quantitative real-time PCR) analysis indicated a significant 89-fold reduction in BCH transcript levels in genome-edited potato lines compared to control plants. Spectrophotometry demonstrated a notable increase in beta-carotene levels in genome-edited potato plants, ranging from 0.831 µg/mL FW to 4.236 µg/mL FW, compared to the control plant with the lowest beta-carotene concentration (0.344 µg/mL FW). HPLC analysis further confirmed increased beta-carotene levels in genome-edited potato plants, ranging from 0.11 mg/mL FW to 0.36 mg/mL FW, compared to the unmodified control plant with a minimum beta-carotene value of 0.09 mg/mL. Our results revealed that the multiplexed CRISPR-Cas9 approach targeting the BCH gene results in enhanced beta-carotene contents in potato tubers.

Solanum tuberosum↗

PubMed2006

Characterization of pFMBL1, a small cryptic plasmid isolated from Leuconostoc mesenteroides SY2.

A 4661bp cryptic plasmid, pFMBL1, was isolated from Leuconostoc mesenteroides SY2, an isolate from Kimchi, and characterized. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. orf2 was 453bp in size and its translation product had 58% identity with a putative protein possibly involved in the replication of pTXL1, a cryptic plasmid from L. mesenteroides ssp. mesenteroides Y110. RNA transcript from orf2 was detected but not from orf1 or intergenic region. Minimum 3.5kb fragment encompassing orf1 and orf2 was required for the replication of pFMBL1 and employed for the construction of Escherichia coli-Leuconostoc shuttle vector, pSJ33E. L. mesenteroides SY1 (another Kimchi isolate), Leuconostoc ssp., and Lactobacillus brevis were successfully transformed with pSJ33E, and the transformation efficiencies were ranged between 1.1x10(1) and 4x10(5)transformants/microg DNA. No single-stranded DNA intermediate was detected from L. mesenteroides SY1 cells harboring pSJ33E, indicating that pFMBL1 probably replicated via theta-type mechanism. pSJ33E was stably maintained in L. mesenteroides SY1 in the absence of erythromycin (Em, 5 microg/ml) and after 1 month of daily subculturing in MRS broth without selective pressure, three percent of cells still retained pSJ33E.

Base Pairing↗

PubMed2007

Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

Amino Acid Sequence↗

PubMed2004

Isolation and characterization of goat ovarian aromatase cDNA: assessment of the activity using an intact cell system and placental expression.

Goat ovarian follicles produce estrone and estradiol from androgens. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase, but little information about this key enzyme is available in the goat. We report here for the first time the cDNA sequence of the goat ovarian aromatase, the activity of the enzyme in a cell system, and its expression in the term goat placenta. A cDNA library from goat ovarian poly(A)+ RNA was constructed. Human aromatase cDNA was selected as probe to screen the library; several clones were isolated, but none was complete. The longest clone was 3.1 kb long, but it lacked the sequence coding for a few amino acids in the NH(2)-terminal. To obtain the missing sequence, we performed reverse amplification of the cDNA end (RACE). Sequence analysis indicated that goat aromatase possessed a very long 3'-untranslated region ( approximately 1790 bp), and a polyadenylation signal (AATAAA) located at position 3320 downstream from the ATG start codon. The coding region of goat cDNA was inserted in an expression vector and transfected into HEK-293 cells that were cultured in presence of [14C]-androstenedione, steroids extracted and further separated by TLC. The transfected cells efficiently transformed [14C]-androstenedione into estrone. This activity was inhibited by 4-hydroxyandrostenedione. We also investigated the presence of mRNA for P450 aromatase in the goat placenta, using reverse transcription-polymerase chain reaction (RT-PCR) and primers derived from the cDNA ovarian sequence and confirmed the expression of the mRNA in term placenta.

Amino Acid Sequence↗

PubMed2005

Prooxidant activity of resveratrol in the presence of copper ions: mutagenicity in plasmid DNA.

Resveratrol, a polyphenolic compound of plant origin, has been of much interest to researchers because of its anticancer and cardiovascular properties. Although antioxidant action of this compound is believed responsible for its reported properties, it has also been shown to exhibit prooxidant properties, especially in the presence of copper ions. Here we report the mutagenicity of resveratrol in plasmid DNA. Plasmid bluescript SK(+) DNA was treated with increasing concentrations of resveratrol in the presence and absence of copper ions, transformed into competent DH5alpha cells and sequenced. We looked for mutations caused by resveratrol treatment by comparing the sequences of treated plasmids versus control (untreated plasmid). The results show a decrease in the transformation efficiency of the plasmid after resveratrol treatment, and although all types of mutations were recorded, point mutations (deletions/substitutions) were found to be the predominant ones. Resveratrol alone resulted in deletion of mainly guanine bases. Since copper ions are known to be found in the nucleus, bound to guanine bases in chromatin, our results suggest mobilization of such endogenous copper by resveratrol resulting in prooxidant DNA cleavage at the site. Concentration of copper is reported to be elevated in various malignancies and the present studies might explain the reported anticancer activity of resveratrol in various cancer cell lines.

Antineoplastic Agents, Phytogenic↗