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At least 19 recordsLinked to original sources
PubMed1996

Catalytic mechanism of enterococcal kanamycin kinase (APH(3')-IIIa): viscosity, thio, and solvent isotope effects support a Theorell-Chance mechanism.

Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the production of enzymes which covalently modify these drugs. The Enterococci and Staphylococci produce an ATP-dependent kinase, APH(3')-IIIa, which phosphorylates such antibiotics as kanamycin, amikacin, and neomycin, and this enzyme shows a Theorell-Chance kinetic mechanism by traditional product and analogue inhibitor analysis and by the alternative substrate diagnostic [McKay, G. A., & Wright, G. D. (1995) J. Biol. Chem. 270, 24686-24692]. We report that the APH(3')-IIIa exhibits small solvent (VH/VD approximately equal to 1.50) and thio effects (VATP/VATP gamma S = 2) indicating hydroxyl group deprotonation and nucleophilic attack on ATP do not significantly contribute to the overall steady-state rate. The enzymatic rates were determined with the viscogens PEG 8000, glycerol, and sucrose, and these experiments demonstrate that ATP binding and ADP release are diffusion controlled and that ADP release is solely rate limiting for APH(3')-IIIa. In addition, the slope of V/K for ATP vs relative viscosity is greater than the theoretical limit of 1, suggesting a possible enzyme conformational change upon binding of ATP. This new experimental evidence supports a Theorell-Chance mechanism for APH(3')-IIIa.

Adenosine Diphosphate↗
PubMed2005

A method for the construction of in frame substitutions in operons: deletion of the essential Escherichia coli holB gene coding for a subunit of the DNA polymerase III holoenzyme.

To investigate the putative five-gene operon at 24.9 min on the Escherichia coli genome, which comprises the genes pabC, yceG, tmk, holB and ycfH, a method for the construction of an in frame deletion strain of the essential E. coli holB gene was developed. HolB, also referred to as delta prime or delta', is a subunit of the DNA polymerase III (Pol III) holoenzyme. The holB gene was replaced by the kanamycin resistance gene kka1, coding for amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene was expressed under the control of the promoter(s) of the putative five-gene operon. The holB gene is essential for bacterial growth and the deletion of holB exhibits no polar effects on the adjacent genes tmk or ycfH in terms of cell viability. The method of the holB null construction presented in this work allows for a simplified studying of interactions between the different subunits of DNA polymerase III.

Bacteriophage P1↗
PubMed2006

Construction and complementation of in-frame deletions of the essential Escherichia coli thymidylate kinase gene.

This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3'-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.

Bacteriophage T4↗
PubMed1998

Expression of the membrane protein glycophorin A as a fusion with the antibiotic resistance protein neomycin phosphotransferase II.

The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5' end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Escherichia coli as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment. Neomycin resistance was directly correlated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step. The amount of GPA-NPT produced was further increased by weakening the specific NPT enzymatic activity via site-directed mutagenesis. Detection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody. This approach may be also applicable to other membrane proteins.

Animals↗
PubMed1989

Stable co-transformation of maize protoplasts with gusA and neo genes.

An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 x BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing beta-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 x 10(-4) (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

DNA↗
PubMed1990

Hybrid genes in the analysis of transformation conditions. 3. Temporal/spatial fate of NPTII gene integration, its inheritance and factors affecting these processes in Nicotiana plumbaginifolia.

Freshly isolated haploid mesophyll protoplasts of Nicotiana plumbaginifolia were transformed for kanamycin resistance. In 38% of the 224 transformants analysed, transmission of the NPTII gene occurred as a homozygous trait, while 62% of the transformants were heterozygous for the trait. In the first case, the foreign DNA integration predominantly (95%) resulted in monogenic inheritance. The second group was characterized by a significant (46%) proportion of multiple insertions. However, there was no clear-cut difference in the integration pattern between the two groups. Furthermore, transformation rates were increased by 4- to 10-fold when transformed diploid protoplasts were treated with UV light or with 3-aminobenzamide. The number of insertion sites was also increased by these treatments. These results shed further light on the fate of the foreign DNA in transformed plants and on means to control or manipulate the integration event(s).

Benzamides↗
PubMed1994

A 6' gentamicin acetyltransferase gene allows effective selection of tobacco transformants using kanamycin as a substrate.

6' gentamicin acetyltransferases detoxify aminoglycoside antibiotics containing a 6' amino group. We tested whether a 6' gentamicin acetyltransferase gene (6' gat) of Shigella sp. is suitable as selectable gene in plant transformation using kanamycin (Km) as a substrate. A comparative transformation experiment using Nicotiana tabacum SR1 protoplasts showed that 6' gat is as effective for selection of transformants as the commonly used neomycin phosphotransferase II (nptII). In stably transformed plants we detected moderate levels of the 6' gat mRNA. An enzymatic assay was developed with which the acetyltransferase activity of the protein is easily demonstrated.

Acetyltransferases↗
PubMed1994

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene.

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 micrograms/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 micrograms/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.

Cells, Cultured↗
PubMed1992

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens.

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

Agrobacterium tumefaciens↗
PubMed1991

Direct regeneration of transformed shoots in Brassica napus from hypocotyl infections with Agrobacterium rhizogenes.

Genetically transformed root clones of rapeseed (Brassica napus) were obtained after in vitro infection of excised hypocotyl segments with a wild type strain of Agrobacterium rhizogenes and two strains of A. rhizogenes harbouring kanamycin resistance. The ability of hairy root formation was affected by light and was highly dependent on the location of the infection site at the hypocotyl. Inoculation of decapitated hypocotyls with an intact root system gave rise to direct shoot formation from the site of inoculation. Histological sections showed that several meristems were initiated at the inoculation site. Root and shoot clones were isolated and subcultured axenically in hormone-free liquid MS medium. Identification of transformed root and shoot clones was based on opine assays. Further selection was carried out in kanamycin-enriched medium. All opine-positive root clones showed NPT II (neomycin phosphotransferase) activity. Nearly half of the shoot clones expressed a strong NPT II activity while the rest gave a weak or no NPT II response.

Brassica↗
PubMed1993

A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots.

There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium. Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium. We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues. The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation. A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus. Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots. These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.

Agrobacterium tumefaciens↗
PubMed1990

Site-directed recombination in the genome of transgenic tobacco.

The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%-100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.

Blotting, Northern↗
PubMed1990

Cauliflower mosaic virus P35S promoter activity in Escherichia coli.

We present evidence that the cauliflower mosaic virus promoter P35S can direct expression of the bacterial neomycin phosphotransferase II (NPTII) gene in Escherichia coli. Transcription is initiated at several sites, the major one being located approximately 315 bases upstream of the plant start site. The nucleotide sequence directly preceding this start site is strongly homologous to the prokaryotic promoter consensus sequence. Thus constructs designed for introduction into plants can be expressed in E. coli.

Base Sequence↗
PubMed1993

Kanamycin resistance as a selectable marker for plastid transformation in tobacco.

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 micrograms/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 micrograms/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10,000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.

Base Sequence↗
PubMed1990

G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae.

Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36-48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.

Amino Acid Sequence↗
PubMed1990

Recognition efficiency of Dicotyledoneae-specific promoter and RNA processing signals in rice.

Heterologous gene expression experiments have shown that genes of Monocotyledoneae are often not transcribed in Dicotyledoneae, or produce pre-mRNA that is inefficiently or aberrantly processed. It is however not known how correctly and efficiently dicotyledon-specific gene expression signals are recognized in cells of Monocotyledoneae. Here we address this question using tobacco (Nicotiana tabacum) and rice (Oryza sativa) protoplasts transformed with the same hybrid gene constructs. Constructs including the nptII protein coding sequence fused to Cauliflower Mosaic Virus (CaMV) promoter and polyadenylation signals were used to obtain stably transformed cell lines of tobacco and rice. In one of the constructs the nptII coding region is interrupted by a modified intron-3 sequence from the soybean phaseolin gene. Although the mean number of hybrid gene copies integrated into the rice genome was on average 5- to 10-fold higher than in tobacco, the steady-state transcript level was 3 times lower. A lower level of transcript was also observed in transient expression experiments. The amount of the mature mRNA was not influenced by the presence of the intron. The phaseolin intron was processed in rice with high efficiency and an accuracy indistinguishable from that seen in tobacco.

Base Sequence↗
PubMed1989

Bleomycin-kanamycin resistance as a marker of the presence of transposon Tn5 in clinical strains of Escherichia coli.

The aminoglycoside modifying enzyme aminoglycoside 3'-phosphotransferase II (APH(3')II) is encoded for on transposon Tn5 by the aphA gene, in the same operon as the ble gene determining bleomycin resistance. To document this linkage 82 kanamycin-resistant Escherichia coli strains of clinical origin were studied; all 18 isolates presenting bleomycin-kanamycin resistance were shown by an enzymatic assay to produce APH(3')II, and the presence of Tn5 was demonstrated by gene hybridization. Similarly, bleomycin-kanamycin resistance was shown to be linked to APH(3')II production in Salmonella spp. The epidemiology of strains with Tn5-encoded APH(3')II may thus be studied, at least in Escherichia coli, by a simple diffusion test using bleomycin and kanamycin discs.

Bleomycin↗
PubMed1990

Evolutionary origin of aminoglycoside phosphotransferase resistance genes.

The protein sequences of seven 3'-aminoglycoside phosphotransferases falling into the six identified types and three 6'-aminoglycoside phosphotransferases were analyzed to give a rooted phylogenetic tree. This tree supports the origin of these groups of enzymes in an ancestor closely related to the actinomycetes, and that horizontal transfer of the resistance genes occurred, possibly via transposons. The implications for genetic engineering of a novel antibiotic are discussed.

Actinomycetales↗