Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Transformation efficiency”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,081 records · Page 60Linked to original sources
PubMed1990

An E mu-v-abl transgene elicits plasmacytomas in concert with an activated myc gene.

To clarify how the v-abl oncogene of Abelson murine leukemia virus contributes to lymphoid tumorigenesis, we introduced the gene linked to an immunoglobulin heavy chain enhancer (E mu) into the mouse germline. Although lymphoid development was not detectably affected in young E mu-v-abl mice, three transgenic lines shared a high predisposition to develop clonal plasmacytomas that secreted IgA or IgG. The unexpected absence of pre-B lymphomas suggests that Abelson virus generates such tumors by infecting an early lymphoid progenitor cell that has not yet activated the heavy chain enhancer. Most plasmacytomas bore a rearranged c-myc gene, apparently as a result of spontaneous translocation to the Igh locus. Moreover, progeny of a cross with analogous E mu-myc mice rapidly developed oligoclonal plasmacytomas. Thus, the collusion of v-abl with c-myc is stage specific, efficiently transforming plasma cells but not pre-B cells or B cells.

Abelson murine leukemia virus↗
PubMed1995

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.

3T3 Cells↗
PubMed2005

Isolation and structural analysis of efficient autonomously replicating sequences (ARSs) of the yeast Candida utilis.

The industrially important yeast Candida utilis is widely used in production of food and medical materials, but its host-vector system has not been well developed. We screened for compact and efficient ARSs to construct practically useful vectors. The C. utilis strain AHU3053 was found to be efficiently transformed by the conventional lithium acetate method and was used as the host. The C. utilis IAM4264 genomic library was constructed by inserting the partial Sau3AI digests in pRI51, which has a kanMX gene expressible in C. utilis. By examining 98 C. utilis G418-resistant transformants, five plasmids had the highest ARS activity. By trimming of the inserts, the 1490 and 552 bp fragments with transformation activity of over 10(3)/microg DNA were obtained from ARS3 and ARS4, respectively. Although several sequences identical to S. cerevisiae ARS consensus sequences (ACSs) were found in ARS3 and ARS4, our deletion analysis indicated that these were not essential for the activity. Because the minimal functional ARS fragment was also several-fold larger than that of S. cerevisiae, the C. utilis ARSs have some unique characteristics resembling the Sz. pombe ARSs. These ARSs were functional in other C. utilis strains tested and useful for constructing practical vectors.

Base Sequence↗
PubMedDate not supplied

A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors.

We describe a set of replicative, integrative and single-stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha-complementation. The nucleotide sequence of each of them is completely known.

Base Sequence↗
PubMed1994

Yeast kar1 mutants provide an effective method for YAC transfer to new hosts.

Yeast artificial chromosome (YAC) clones propagate large segments of exogenous DNA in a host organism with well-developed classical and molecular genetics. Most extant YAC clones are from libraries created in a single yeast host (AB1380). The application of techniques allowing the manipulation and/or restructuring of these cloned DNA segments often requires a change in the yeast genetic background to introduce desirable genetic markers. Transfer methods in current use require extremely high yeast transformation efficiencies or require access to equipment for yeast tetrad analysis. We have developed an alternative method for moving YAC clones from one yeast strain to another, taking advantage of the properties of kar1 mutants altered in a gene required for normal karyogamy (nuclear fusion) during mating. Transfer by this method requires generally accessible methods, including yeast cell culture, replica plating, and pulsed-field gel electrophoresis. We present data demonstrating efficient transfer of nine different YACs from their original host (AB1380) to a kar1 recipient strain (YPH925) with genetic markers that facilitate the use of existing homologous recombination-based modification methods. The enhanced ability to transfer clones to this new host will accelerate the pace of refinement and fine-structure mapping of the YAC contigs currently under construction and facilitate gene manipulation on YACs for subsequent functional analysis.

Chromosomes, Artificial, Yeast↗
PubMed1997

Characterization of the mycobacteriophage L5 attachment site, attP.

Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome. Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein. DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange. Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration. We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria.

Bacteriophages↗
PubMed1995

Competition between parental and recombinant plasmids affects the measure of recombination frequencies.

Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells"). Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid. Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements. To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid. We used pBR322-derived plasmids which carry or lack the replication terminator TerB; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid. The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling. The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells. Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells. This effect can be as high as several orders of magnitude.

Base Sequence↗
PubMed1993

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene.

We have successfully transferred and expressed a reporter gene driven by an alpha-amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10-12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of beta-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice alpha-amylase gene (alpha Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice alpha-amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.

Agrobacterium tumefaciens↗
PubMed1993

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize.

A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the beta-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the -208 to -46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to -389 bp from ATG) promoter of wheat, alpha-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10-20 resistant calli, or GUS-expressing colonies after treatment of 10(6) protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80-90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.

Aminobutyrates↗
PubMed1992

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.

Amino Acid Sequence↗
PubMed1983

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells.

Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk- cells as well as E. coli tk- cells to the Tk+ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk- mutant of E. coli C600.

Escherichia coli↗
PubMed1981

Regulation of plasmid DNA synthesis: isolation and characterization of copy number mutants of miniR6-5 and miniF plasmids.

Copy number (Cop) mutants of miniR6-5 and miniF plasmid derivatives containing a beta-lactamase gene were isolated by selection for increased ampicillin-resistance. Mutants which exhibited an increased copy number and a reduced incompatibility response as compared to the respective parent mini-plasmid were obtained from both miniR6-5 and miniF. Characterization of these mutant plasmids has provided the first description of the replicative properties of miniF Cop mutants, and has also facilitated a comparison of plasmids representing the IncFI and IncFII incompatibility groups. Cop mutants from these groups differed in several respects: (i) MiniF Cop mutants were considerably more difficult to obtain and showed a markedly lower transforming efficiency than the corresponding miniR6-5 mutants. (ii) MiniR6-5 Cop mutants were stably maintained in a polA1 strain without selective pressure, whereas miniF Cop mutants severely reduced the viability of this host. (iii) MiniR6-5 replication stopped within a few minutes after inhibition of protein synthesis, whereas miniF replication continued at a declining rate for about one hour in the presence of chloramphenicol. (iv) In contrast to miniR6-5 replication, miniF DNA synthesis was blocked faster by rifampicin than by chloramphenicol. (v) The copy number of miniR6-5 plasmids (but not of miniF) was reduced by about 50% in an rnc strain deficient in RNAase III.

Ampicillin↗
PubMed1986

Controlled gene expression utilising lambda phage regulatory signals in a cyanobacterium host.

This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.

Acetyltransferases↗
PubMed1979

A hex mutant of Haemophilus influenzae.

A mutant of Haemophilus influenzae which does not discriminate between low efficiency (LE) and high efficiency (HE) markers has been isolated. The mutant does not differ wild type in its sensitivity to ultraviolet radiation, methyl methanesulfonate (MMS) mitomycin C, and nitrous acid. Spontaneous mutation frequencies for three loci studied are 10- to 30-fold higher in the mutant than in the wild type strain. Low- and high-efficiency transforming markers are equally UV-resistant when assayed on this mutant. This mutant is thus similar to the hex mutant of Streptococcus pneumoniae.

DNA Repair↗
PubMed1993

Synthesis of oligosaccharides with oligoethylene glycol spacers and their conversion into glycoconjugates using N,N,N',N'-tetramethyl(succinimido)uronium tetrafluoroborate as coupling reagent.

Glycosides of glucose and lactose with di- and tetraethylene glycols, transformed into bifunctional (alcohol, ester) spacer molecules, have been synthesized. After deprotection, these spacer glycosides, containing a free carboxyl group, have been transformed efficiently into glycoconjugates using N,N,N',N'-tetramethyl(succinimido)uronium tetrafluoroborate (TSTU) for the formation of an active ester.

Ethylene Glycols↗
PubMedDate not supplied

Genetic organization of Acetobacter for acetic acid fermentation.

Plasmid vectors for the acetic acid-producing strains of Acetobacter and Gluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochrome c, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene of A. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of an A. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of the Acetobacter strains was developed and applied to improve their properties.

Acetates↗
PubMed1996

Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants.

A set of plasmids has been constructed utilizing the promoter, 5' untranslated exon, and first intron of the maize ubiquitin (Ubi-1) gene to drive expression of protein coding sequences of choice. Plasmids containing chimaeric genes for ubiquitin-luciferase (Ubi-Luc), ubiquitin-beta-glucuronidase (Ubi-GUS), and ubiquitin-phosphinothricin acetyl transferase (Ubi-bar) have been generated, as well as a construct containing chimaeric genes for both Ubi-GUS and Ubi-bar in a single plasmid. Another construct was generated to allow cloning of protein coding sequences of choice on Bam HI and Bam HI-compatible restriction fragments downstream of the Ubi-1 gene fragment. Because the Ubi-1 promoter has been shown to be highly active in monocots, these constructs may be useful for generating high-level gene expression of selectable markers to facilitate efficient transformation of monocots, to drive expression of reference reporter genes in studies of gene expression, and to provide expression of biotechnologically important protein products in transgenic plants.

Base Sequence↗
PubMed2005

QTLs controlling the production of transgenic and adventitious roots in Brassica oleracea following treatment with Agrobacterium rhizogenes.

Brassica oleracea can be genetically engineered using Agrobacterium rhizogenes. The initial stage of this process is the production of transgenic ('hairy') roots; shoots are subsequently regenerated from these roots. Previous work using gus and gfp reporter genes has shown that genotypes of B. oleracea vary in their performance for transgenic root production. Quantitative trait loci (QTLs) controlling this trait have been located in one mapping population. The current study provides evidence that performance for transgenic root production is associated with performance for adventitious (non-transgenic) root production in B. oleracea across a second mapping population. This is shown by regression analyses between performance for the two traits and the demonstration that QTLs controlling the two traits map to the same positions within the genome. Since the rate of adventitious root production does not differ significantly in the presence and absence of A. rhizogenes, there is no evidence that the expression of Agrobacterium genes induces adventitious root production. It is apparent that genotypes exhibiting high adventitious root production in the absence of A. rhizogenes will also tend to show high transgenic root production, thereby allowing the selection of lines that are more efficiently transformed.

Brassica↗