Electric field-mediated DNA encapsulation into large liposomes.

Large, ethidium bromide-loaded liposomes electrically pulsed in the presence of externally added DNA display the bright fluorescence of DNA-ethidium bromide complexes. Sonication of these liposomes increases the fluorescence of trapped DNA-ethidium bromide complexes by no more than about 40%. These results are thus in agreement with a mechanism involving electropores for DNA uptake but do not support an alternative mechanism, invoking invagination and pinching-off of the lipid bilayer, through which internalized DNA is shielded from the liposome contents.

Coat protein-mediated resistance to pea enation mosaic virus in transgenic Pisum sativum L.

Pea (Pisum sativum L.) plants were transformed in planta by injection/electroporation of axillary meristems with a chimeric pea enation mosaic virus (PEMV) coat protein gene contruct. R1 progenies of these plants were shown to harbor the transgene by polymerase chain reaction (PCR) and genomic Southern analysis, while transgene expression was demonstrated by western blot analysis. Transgenic R2, R3 and R4 plants displayed delayed or transient PEMV multiplication and attenuated symptoms as compared to control inoculated individuals.

Half-embryo cocultivation technique for estimating the susceptibility of pea (Pisum sativum L.) and lentil (Lens culinaris Medik.) cultivars to Agrobacterium tumefaciens.

Longitudinally sliced embryonic axes from pea and lentil mature seeds cocultivated with A. tumefaciens carrying a gus reporter gene in its T-DNA provided a convenient means to evaluate the efficiency of gene transfer to tissues in different cultivars and cocultivation conditions. Use of this technique demonstrated wide variation in susceptibility to Agrobacterium among several pea and lentil commercial genotypes.

Gene transfer by electroporation.

Electroporation of cells in the presence of DNA is widely used for the introduction of transgenes either stably or transiently into bacterial, fungal, animal, and plant cells. A review of the literature shows that electroporation parameters are often reported in an incomplete or incorrect manner, forcing researchers to rely too much on a purely empirical trial and error approach. The goal of this article is to provide the reader with an understanding of electrical circuits used in electroporation experiments as well as physical and biological aspects of the electroporation process itself. Further, a simple paradigm is provided which unites all electroporation parameters. This article should be particularly useful to those new to the technique.

Transgenic grain legumes obtained by in planta electroporation-mediated gene transfer.

Electroporation-mediated gene transfer into intact plant tissues was demonstrated in pea, cowpea, lentil, and soybean plants. Transient expression of a chimeric gus reporter gene was used to monitor the uptake and expression of the introduced DNA in electroporated nodal axillary buds in vivo. The branches that grew out of the nodal meristems were chimeric and expressed the introduced gene up to 20 d after electroporation. Transgenic R1 pea, lentil, and cowpea plants were recovered from seeds originating on these chimeric branches as shown by Southern blot hybridization and GUS expression. Transgenic R2 soybean and lentil plants were also obtained. Segregation ratios in these populations showed a strong bias against transgene presence or expression.

Electroporation-mediated gene transfer into intact nodal meristems in planta. Generating transgenic plants without in vitro tissue culture.

Transient expression and stable integration and expression of transgenes were observed in the tissues and offspring of certain leguminous plants after electroporation of DNA into intact nodal meristems in planta. The method described in this article thus allows the study of transgene expression in tissues differentiating from meristematic cells present in the treated buds. In addition, transgenic plants can be recovered in the offspring of electroporated individuals. Therefore, this technique allows the production of transgenic leguminous plants without the need for in vitro tissue culture, often a major hurdle with this family.

Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos.

Cowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing ß-glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and Lipofectin(TM) increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.

Transient expression and histological localization of a gus chimeric gene after direct transfer to mature cowpea embryos.

Transient expression of a chimeric gus reporter gene was demonstrated in cowpea embryonic cells after direct gene transfer. This report shows that seed-derived embryos express the reporter gene at high frequency after passive or electroporation-mediated DNA transfer.

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4.

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.

Stable transformation of eggplant (Solanum melongena L.) by cocultivation of tissues withAgrobacterium tumefaciens carrying a binary plasmid vector.

Stable transformation of eggplant to kanamycin resistance was obtained by cocultivation of cotyledonary and young leaves with the hypervirulent, fully oncogenicAgrobacterium tumefaciens strain A281 carrying plasmid pGA472. No transformation was observed when using the disarmedA. tumefaciens LBA4404 strain carrying pGA472 or when using either strain for cocultivation with eggplant suspension cells.The NPTII enzyme and DNA dot blot assays performed on callus cells growing in the presence of kanamycin indicated both the presence and expression of the foreign gene. The highest proportion of transformed explants was obtained from intact cotyledonary leaf pieces while the highest NPTII enzyme specific activity was detected in callus cells originating from superficially wounded cotyledonary leaf pieces. Kanamycin-resistant plantlets were regenerated after six months in culture.

Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4).

The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid. tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway. The tfdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, tfdB to tfdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W.R. Streber, K. N. Timmis, and M. H. Zenk, J. Bacteriol. 169:2950-2955, 1987). A second, dissimilar locus on pJP4, tfdAII, has been observed which encodes 2,4-D monooxygenase activity. Gas chromatographic analysis of the 2,4-D metabolites of A. eutrophus harboring pJP4 or subclones thereof localized tfdAII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes tfdBCDEF. This fragment was further characterized in Escherichia coli by deletion and subcloning analysis. A region of 2.5 kilobases, adjacent to tfdC, enabled E. coli extracts to degrade 2,4-D to 2,4-dichlorophenol. Hybridization under low-stringency conditions was observed between tfdA and tfdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.

A plasmid-based method to quantitate homologous recombination frequencies in gram-negative bacteria.

A method is described which enables quantitative evaluation of the ability of gram-negative bacterial cells to perform homologous recombination between DNA molecules. This method is particularly useful in cases where the stringency of rec mutations is to be determined. The procedure is based on a wide-host-range vector (pRK404) in which two unequally truncated and overlapping fragments of the neo gene were cloned. When introduced into gram-negative bacteria either by transformation or by conjugation, molecules of this plasmid, pBX404-7, undergo unequal crossing-over leading to the restoration of a functional neo gene. The stringency of putative rec mutations can thus be determined by measuring the frequency at which kanamycin-resistant colonies appear in bacterial strains harboring pBX404-7.

Study of plant protoplast-bacterial spheroplast and cell interactions by flow cytometry.

The analyzer component of the Becton-Dickinson Fluorescence Activated Cell Sorter (FACS) 420 was used to investigate the binding of bacterial spheroplasts and cells to plant protoplasts. Several binding parameters (incubation time, protoplast density and spheroplast/protoplast ratio were determined.

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens.

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype.

Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides.

A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress. The minicells so prepared are metabolically active as indicated by the in vivo incorporation of [35S]methionine into plasmid-coded polypeptides.

Targeting of large liposomes with lectins increases their binding to plant protoplasts.

Soybean agglutinin, peanut agglutinin, and concanavalin A were covalently bound by condensation reaction to gangliosides and ceramides incorporated within the bilayer of multilamellar and unilamellar liposomes. These modified liposomes had a much higher affinity for carrot and tobacco protoplasts except when concanavalin A was used.In addition, soybean agglutinin and concanavalin A were attached by ligand-specific binding to liposomes containing cholesterol molecules derivatized with each lectin-specific sugar. This procedure allowed efficient crosslinking of liposomes to protoplasts. The same effect was achieved with soybean agglutinin and peanut agglutinin when derivatized cholesterol was replaced by gangliosides. The implications of these findings for the liposome-mediated nucleic acid transfer into protoplasts are discussed.

Infection of cowpea mesophyll protoplasts with cowpea chlorotic mottle virus (CCMV) RNA encapsulated in large liposomes.

It has been demonstrated that cowpea chlorotic mottle virus RNA encapsulated in phosphatidyl serine/cholesterol reverse evaporation vesicles (REV) could infect cowpea mesophyll protoplasts under conditions known to enhance liposome-protoplast interactions. Positively charged phosphatidylcholine/stearylamine multilamellar liposomes did not deliver functional CCMV RNA despite their very high nucleic acid trapping capacity and their high affinity for protoplasts.