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At least 181 records · Page 10Linked to original sources
PubMed1998

Practical synthesis of androgen: the efficient transformation of 17-oxo group to 17 alpha-hydroxy group.

The present report describes the improved transformation of the 17-oxo group in 3 beta-acetoxy-5 alpha-androstan-17-one to a 17 alpha-hydroxy group. A mixture of 17 alpha-acetoxy and 16-ene compounds, which are usually produced by the standard synthetic route, were treated with peracetic acid (epoxidation of the 16-ene compound) and then sodium borohydride-sodium hydroxide (reduction-hydrolysis) to give the desired 17 alpha-hydroxy compound in much better yield than that in previous reports. Recrystallization of the crude product with cyclohexane-methanol gave the pure compound in 54% yield (total yield from starting ketone).

Androgens↗
PubMed2000

Efficient transformation of Dictyostelium discoideum with a particle inflow gun.

We report experiments to transform Dictyostelium discoideum using a simple home-made particle gun. Stable transformants were obtained at frequencies of up to 2500 clones/microg DNA. This is five times more than we achieve with the same vector using electroporation protocols. We also show that the particle inflow gun can be used for analysis of developmentally regulated gene expression in a transient assay.

Animals↗
PubMed2005

PdCl2-catalyzed efficient transformation of propargylic amines to (E)-alpha-chloroalkylidene-beta-lactams.

[reaction: see text] The PdCl2-catalyzed cyclocarbonylation reaction of propargylic amines with CuCl2 and benzoquinone afforded (E)-alpha-chloroalkylidene-beta-lactams in moderate to good yields. The formation of the corresponding Z-isomers or five-membered products was not observed. The reaction of the readily available optically active propargylic amines provides a convenient synthesis of the corresponding (E)-alpha-chloroalkylidene-beta-lactams with high ee values. The structure and the stereochemistry of the products were established by the X-ray single-crystal diffraction study of (E)-6d and (E)-6e, which indicates that the stereoselectivity in this reaction is different from what was observed with propargylic alcohols. A rationale for this reaction was proposed.

Journal Article↗
PubMed1997

High-efficiency transformation of Arabidopsis thaliana with a selectable marker gene regulated by the T-DNA 1' promoter.

Two selectable marker genes harbouring the bar coding region but differing in their promoters were compared in an Arabidopsis thaliana transformation assay using in planta infiltration with Agrobacterium tumefaciens. Surprisingly, in four Arabidopsis ecotypes examined, the 1' promoter from the right T-DNA was superior to the most commonly used 35S promoter of cauliflower mosaic virus (CaMV). The ecotype Wassilewskija gave the highest transformation frequencies, with an average of between 5.3 and 6.3% of the seedlings subjected to the selection. This is approximately 30-fold higher than previously reported results. Analysis of T-DNA integration patterns in single transformed plants or pooled populations revealed independent T-DNA integration events in each case. Results show that the 1' promoter is an attractive alternative to the 35S promoter for the generation of T-DNA insertion lines. The 1' promoter may be especially beneficial for the secondary transformation of transgenic strains containing the 35S promoter to exclude homology-mediated gene silencing.

Agrobacterium tumefaciens↗
PubMed1997

Highly mismatched molecules resembling recombination intermediates efficiently transform mismatch repair proficient Escherichia coli.

The ability of related DNAs to undergo recombination decreases with increased sequence divergence. Mismatch repair has been proposed to be a key factor in preventing homeologous recombination; however, the contribution of mismatch repair is not universal. Although mismatch repair has been proposed to act by preventing strand exchange and/or inactivating multiply mismatched heteroduplexes, there has been no systematic study to determine at what step(s) in recombination mismatch repair acts in vivo. Since heteroduplex is a commonly proposed intermediate in many models of recombination, we have investigated the consequences of mismatch repair on plasmids that are multiply mismatched in heteroduplex structures that are similar to those that might arise during recombination. Plasmids containing multiply mismatched regions were transformed into wild-type and Mut+ Escherichia coli mutants. There was only a 30-40% reduction in transformation of Mut+ as compared to mutS and mutL strains for DNAs containing an 18% mismatched heteroduplex. The products obtained from mutS hosts differed from those obtained from Mut+ hosts in that there were many more colonies containing mixtures of two plasmids, due to survival of both strands of the heteroduplex. There were nearly 10 times more recombinants obtained from the mutS as compared to the wild-type host. Based on these results and those from other studies with E. coli and yeast, we propose that the prevention of recombination between highly diverged DNAs may be at a step earlier than heteroduplex formation.

DNA Repair↗
PubMed2006

Common plantain. A collection of expressed sequence tags from vascular tissue and a simple and efficient transformation method.

The vascular tissue of higher plants consists of specialized cells that differ from all other cells with respect to their shape and size, their organellar composition, their extracellular matrix, the type of their plasmodesmata, and their physiological functions. Intact and pure vascular tissue can be isolated easily and rapidly from leaf blades of common plantain (Plantago major), a plant that has been used repeatedly for molecular studies of phloem transport. Here, we present a transcriptome analysis based on 5,900 expressed sequence tags (ESTs) and 3,247 independent mRNAs from the Plantago vasculature. The vascular specificity of these ESTs was confirmed by the identification of well-known phloem or xylem marker genes. Moreover, reverse transcription-polymerase chain reaction, macroarray, and northern analyses revealed genes and metabolic pathways that had previously not been described to be vascular specific. Moreover, common plantain transformation was established and used to confirm the vascular specificity of a Plantago promoter-beta-glucuronidase construct in transgenic Plantago plants. Eventually, the applicability and usefulness of the obtained data were also demonstrated for other plant species. Reporter gene constructs generated with promoters from Arabidopsis (Arabidopsis thaliana) homologs of newly identified Plantago vascular ESTs revealed vascular specificity of these genes in Arabidopsis as well. The presented vascular ESTs and the newly developed transformation system represent an important tool for future studies of functional genomics in the common plantain vasculature.

Arabidopsis↗
PubMed1982

Liposomes as gene carriers: efficient transformation of mouse L cells by thymidine kinase gene.

Stable transformation of mouse L cells deficient in thymidine kinase was achieved by liposome-mediated transfer of a recombinant plasmid carrying the thymidine kinase gene. Ten percent of the recipient cells expressed thymidine kinase activity. The transformed phenotype (for example, 200 out of 10(6) cells) was stable under selective and nonselective conditions. The liposome technique is compared with other methods currently used for gene transfer.

Animals↗
PubMed1985

Efficient transformation of Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica.

We used a modified version of the method of Hanahan (D. Hanahan, J. Mol. Biol. 166:557-580, 1983) to transform Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica with the plasmids pBR322, pBR325, and pAT153. The transformation frequency ranged from 1 X 10(2) to 4 X 10(4) colonies per micrograms of plasmid DNA. The nature of these transformants was confirmed by plasmid analysis. ColE1-based plasmids make potentially useful cloning vectors for the study of genes involved in the pathogenesis of this species.

Erwinia↗
PubMed1989

Hybrid genomes of the polyomaviruses JC virus, BK virus, and simian virus 40: identification of sequences important for efficient transformation.

Hybrid viral genomes were used to investigate the influence of specific polyomavirus sequences on the transforming behavior of JC virus (JCV). One set of chimeric DNAs was made by exchanging the regulatory regions between JCV and simian virus 40 (SV40) or JCV and BK virus (BKV). A second set of constructs was produced that expressed hybrid JCV-BKV T proteins under the control of either JCV or BKV regulatory signals. Transformation of Rat 2 cells with the parental and chimeric DNAs indicated that both the JCV regulatory signals and the sequence encoding the amino terminus of T protein contributed to the restricted transforming behavior of this virus. Analysis of the viral proteins in the transformed rat cells indicated that the large T antigens of JCV and BKV were less stable than their SV40 counterpart, that small t protein was produced in JCV transformants, and that the subpopulation of T antigen that forms a stable complex with cellular p53 protein was smaller in JCV-transformed cells than in SV40- or BKV-transformed cells.

Animals↗
PubMed1994

Autophosphorylation is required for high kinase activity and efficient transformation ability of proteins encoded by host range alleles of v-src.

pp60v-src is a nonreceptor protein tyrosine kinase that can transform both chicken and rodent fibroblasts. The src homology 2 (SH2) domain of this protein serves a critical role in the regulation of protein tyrosine kinase activity. The host range proteins pp60v-src-L, which contains a deletion of a highly conserved residue (Phe-172) in the SH2 domain, and pp60v-src-PPP, which contains a change from a Leu to a Phe at amino acid 186 in the SH2 domain, transform chicken but not rat cells and have slightly reduced kinase activity measured in vitro. The data presented here show that these altered proteins require autophosphorylation on Tyr-416 for high kinase activity and transforming ability. In the absence of autophosphorylation, there is a further decrease of at least threefold in in vitro kinase activity relative to the phosphorylated host range parental protein, no morphological transformation, a reduction in anchorage independent growth, and no disruption of the actin cytoskeleton. In addition, these SH2 mutations abolish the ability of the SH2 domain to bind a phosphorylated peptide that corresponds to the autophosphorylation site of pp60src. Thus, like mutant alleles of c-src encoding transformation competent proteins, and unlike v-src, transformation by pp60v-src-F172 delta and pp60v-src-L186F is dependent on phosphorylation of Y-416 for high kinase activity and transformation ability. The dependence of transformation on phosphotyrosine is not a reflection of an intramolecular interaction between the autophosphorylation site and the SH2 domains since purified SH2 domains are incapable of binding phosphorylated autophosphorylation site peptides in vitro.

Alleles↗
PubMed1983

Efficient transformation of Dictyostelium discoideum amoebae.

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.

Anti-Bacterial Agents↗
PubMed1986

High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion.

After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.

DNA Restriction Enzymes↗