Overcoming hybrid incompatibility between Nicotiana africana Merxm. and N. tabacum and development of cytoplasmically male sterile tobacco forms

The incompatibility between the wild species N. africana Merxm. and the cultivated species N. tabacum has been overcome by in vitro techniques. Underdeveloped F₀ seeds, placed on MS medium with supplements, produced plants which upon reaching the stage of anthesis proved to be completely sterile. Female sterility of F₁ hybrids was overcome by applying tissue culture methods. Explants of stem parenchyma were grown in vitro. In every passage investigations were made of their callus production, organogenesis and cell polyploidization. The regenerants showed a great diversity in their morphological and cytological characters. Pollination of the R₁ plants (N. africana × N. tabacum) with N. tabacum produced normally seeded capsules. BC₁ plants were male sterile. The male sterility of the first backcross generation was preserved in BC₂ and BC₃, proving its cytoplasmic origin.     

Bacteriophage P1 site-specific recombination. III. Strand exchange during recombination at lox sites

When hybrid λ-P1 phages containing either loxP or loxR sites are crossed under conditions where only the P1 lox site-specific recombination system is active, most of the crossovers occur in a region of the DNA that contains the lox sites. The remainder of the lox-mediated crossovers (4% in a P × P cross and 32% in a P × R cross) occur close to, but outside of, either loxP or loxR. These latter crossovers are not detected if one of the partners in the cross carries a deletion of loxP. We explain these results by an exchange of strands at lox sites and a migration of the resulting cross-strand junction outside of lox.     

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Seed-specific gene activation mediated by the Cre/lox site-specific recombination system.

The Cre/lox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a beta-glucuronidase marker gene, by removal of a lox-bounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after-Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) beta-phaseolin and the alpha' subunit of soybean (Glycine max) beta-conglycinin, as well as the cauliflower mosaic virus 35S promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. The 35S promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmental staging, and spread progressively throughout the embryo. The timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. Limited gene activation provides a system for cell lineage and developmental analyses.     

Plant regeneration from cell suspension-derived protoplasts of Nicotiana africana

Plants have been regenerated from Nicotiana africana Merxm. protoplasts isolated from cell suspensions. Two different sequences of media were assayed, one usually used to regenerate tobacco mesophyll protoplasts (K3,RMO) the other previously recommended for potato mesophyll protoplast regeneration (W-S-S, ST-1, ST-2, S-3). Only the media for potato protoplasts were efficient for African tobacco plant regeneration. The regeneration efficiency was 6.3 plants per 1000 plated protoplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids

Bacillus subtilis competent cells harboring plasmid pUB110 were transformed by plasmids unable to replicate in this host but carrying segments of pUB110, 260 to 4500 bp long. Recombinants between the incoming and the resident plasmids were found in the transformed cells. Transforming efficiency of the incoming plasmids depended strongly on their molecular form and the length of their region homologous with the resident plasmid. It increased with the fourth to fifth power of that length for monomers having at least 900 bp of homology. Activity of monomers having less than 900 bp homology was too low to be measured in our experiments. Transforming efficiency of dimers was much greater than that of monomers, and varied with the square of the length of the homologous region. These results indicate that dimeric and monomeric plasmid molecules are processed differently during transformation of B. subtilis competent cells.     

Genetic rearrangements of plasmids containing mtDNA inserts: possible poison sequence

Restriction mapping of recombinant plasmids indicated the presence of poison sequence(s) in monkey mtDNA. These plasmids were constructed from a 5.2 K.b. BglII mtDNA fragment and pRSVneo or pdel9 as cloning vectors. The poison sequence(s) caused genetic rearrangement of the vectors' nucleotide sequences. Deletion of the suspected poison sequence(s) from the mtDNA fragment increased the transformation efficiency of the produced recombinant plasmids and conserved the vectors' original nucleotide sequences.     

Genetic transformation of Medicago truncatula using Agrobacterium with genetically modified Ri and disarmed Ti plasmids

Summary Fertile transgenic plants of the annual pasture legume Medicago truncatula were obtained by Agrobacterium-mediated transformation, utilising a disarmed Ti plasmid and a binary vector containing the kanamycin resistance gene under the control of the cauliflower mosaic virus 35S promoter. Factors contributing to the result included an improved plant regeneration protocol and the use of explants from a plant identified as possessing high regeneration capability from tissue culture. Genes present on the T-DNA of the Ri plasmid had a negative effect on somatic embryogenesis. Only tissue inoculated with Agrobacterium strains containing a disarmed Ti plasmid lacking the T-DNA region or a Ri plasmid with an inactivated rol A gene regenerated transgenic plants. Fertile transgenic plants were only obtained with disarmed A. tumefaciens, and the introduced NPT II gene was transmitted to R₁ progeny.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - NPT neomycin phosphotransferase     

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.     

Chromosomal transformation of Escherichia coli recD strains with linearized plasmids.

Wild-type Escherichia coli are resistant to genetic transformation by purified linear DNA, probably in part because of exonuclease activity. We demonstrate that E. coli containing a recD mutation could be easily transformed by linearized plasmids containing a selectable marker. The marker was transferred to the chromosome by homologous recombination, whereas plasmid markers not in the region of homology were lost.     

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate. II. Separation of plasmids by transformation.

Transformational separation of plasmids from R404 plasmid aggregate found in Salmonella enteritidis strain was performed. Three classes of transformants differing in their resistance patterns were isolated. Genetic properties of the transformants suggest that their resistance is determined by single plasmids. Plasmid pCK3 (Tra-ApCbCrSuSm) and pCK4 (Tra-ApCbCrCm) are nonconjugative while plasmid pCG1 (TraApCbCrSuSmTcKmNm) is conjugative. Separation of all plasmids of R404 plasmid aggregate allowed to determine their genetic properties and the manner of conjugational transfer of R404 plasmid aggregate R-determinants.     

Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant.

The mer operon from a strain of Thiobacillus ferrooxidans (C. Inoue, K. Sugawara, and T. Kusano, Mol. Microbiol. 5:2707-2718, 1991) consists of the regulatory gene merR and an operator-promoter region followed by merC and merA structural genes and differs from other known gram-negative mer operons. We have constructed four potential shuttle plasmids composed of a T. ferrooxidans-borne cryptic plasmid, a pUC18 plasmid, and the above-mentioned mer determinant as a selectable marker. Mercury ion-sensitive T. ferrooxidans strains were electroporated with constructed plasmids, and one strain, Y4-3 (of 30 independent strains tested), was found to have a transformation efficiency of 120 to 200 mercury-resistant colonies per microgram of plasmid DNA. This recipient strain was confirmed to be T. ferrooxidans by physiological, morphological, and chemotaxonomical data. The transformants carried a plasmid with no physical rearrangements through 25 passages under no selective pressure. Cell extracts showed mercury ion-dependent NADPH oxidation activity.     

Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.     

Genetic recombination during transformation in Bacillus subtilis: appearance of a deoxyribonucleic acid methylase.

In Bacillus subtilis the ability to take up deoxyribonucleic acid (DNA) and undergo genetic transformation may coincide with the induction of defective phage(s) and the expression of possibly related cryptic genes. A restriction-modification enzyme system appears to be expressed. Targets of the restriction activity on the DNA can be blocked my methylation catalyzed by the methyl transferase. It is shown that cellular DNA becomes progressively methylated and reaches the maxium level during the peak of competency. Deoxycytidine residues of both incoming donor and resident DNA are methylated. The possible participation of these enzymes in recombination and the general role of cryptic genes in inducible functions are discussed.     

Studies on transformation of Escherichia coli with plasmids

Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.     

Site-specific recombination by the bacteriophage P1 lox-Cre system. Cre-mediated synapsis of two lox sites

The bacteriophage P1-encoded recombinase Cre forms a simple DNA-protein complex at the specific recognition site loxP. Furthermore, Cre is able to mediate a synaptic union of two loxP sites. When two loxP sites are on the same linear DNA molecule, Cre binds the two sites together to form a circular protein-DNA complex. These complexes can be resolved into a linear DNA molecule and a closed circular DNA molecule, the end products of site-specific recombination.     

Site-specific recombination between plasmids of Staphylococcus aureus

Anomalous recombination between two similar but nonidentical, naturally occurring penicillinase plasmids, pI258 and pI524, leading to duplication and deletion of the beta-lactamase locus, is described. Physical mapping of these plasmids by heteroduplex and restriction analysis revealed that the beta-lactamase loci were homologous and in inverted orientation with respect to one another and that their respective locations were separated by a short region of homology. This intervening region of homology included one copy of a segment that was repeated on pI524 in inverted orientation at a distance of 2.2 kilobase pairs and contained a recognition sequence for a site-specific, rec-independent recombination function that caused reversible inversion of this segment on pI524. It is proposed that site-specific, intermolecular recombination involving this repeated sequence was responsible for the observed results.