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Genetic transformation was employed to attain exact identification of Eikenella corrodens and Cardiobacterium hominis. The two species appeared both homogeneous and without genetic affinity to each other or to species of Kingella, Neisseria, and Moraxella, as tested for by rather sensitive procedures with streptomycin- and spectinomycin-resistance markers.
Genetic transformation was used in an attempt to subdivide the most prevalent auxotypes of Neisseria gonorrhoeae in local isolates. The large proline requiring (Pro-) group could be divided into two genetic types, as could the less common arginine requiring (Arg-) group. The large arginine, hypoxanthine, and uracil requiring (Arg- Hyp- Ura-) group could not be subdivided by this method. The genetic relation between these and other auxotypes was investigated.
Streptococcus sanguis (serogroups H and W) strains are frequently competent in genetic transformation. In a collection of 18 strains of S. sanguis isolated from blood cultures, 12 strains were spontaneously competent in transformation by streptomycin-resistant DNA, and three strains were induced to competence by the use of culture filtrates containing competence factor (CF). Culture filtrates from spontaneously competent S. sanguis strains only induced competence in transformation of strains of the same species. In addition to the three "constitutional" types of S. sanguis with regard to specificity of transformation previously reported (originally represented by the strain Challis, 13b and NCTC 7863), a possible fourth type, (represented by strain 21452) was found. Further studies are required to ascertain the role of these types. The "constitutional" types are characterized by the range of activity of their CF's. These specificities seem useful in identification of S. sanguis and its separation from S. mitis (mitior). Tests of spontaneously competent strains with culture filtrates from other competent strains showed variable effects on the transformation frequencies in different strains.
Chloroplast genetic transformation is a new way of plant genetic engineering. This paper reviews the construction methods of expression vector used in chloroplast genetic transformation. It contains the homologous recombinant fragments, the chloroplast specific promoter and terminater, the selectable marker genes and the interest genes whose expression in chloroplast have been achieved.
To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.
Plastid genetic transformation has been performed using both the PEG-treatment of protoplasts of somatic hybrids of B. oleracea carrying A. thaliana chloroplasts and the particle bombardment of regenerable calluses of B. napus sv. Westar. The chloroplast transformation vector pCB040 carried resistance (aadA) gene flanked by rapeseed plastid DNA sequences to target its insertion between the trnV-rps7 fragments. Selection of transplastomic cell lines has been performed according to their ability to grow on the medium supplied with spectinomycin and streptomycin in high concentrations. Antibiotic resistant cell lines have been obtained using the both transformation methods. The presence of the aadA gene in the A. thaliana and B. napus plastomes was confirmed by PCR analysis for two cell lines of B. oleracea (+ A. thaliana) and three lines of B. napus.
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This review examines interactions between DNA and soil with an emphasis on the persistence and stability of DNA in soil. The role of DNA in genetic transformation in soil microorganisms will also be discussed. In addition, a postulated mechanism for stabilization and elongation/assembly of primitive genetic material and the role of soil particles, salt concentrations, temperature cycling and crystal formation is examined.
Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer. During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell, within which T-DNA nuclear import is mediated by VirD2 (ref. 3) and VirE2 (ref. 4) and their host cell interactors AtKAP-alpha and VIP1 (ref. 6), whereas its integration is mediated mainly by host cell proteins. The factors involved in the uncoating of T-DNA from its cognate proteins, which occurs before integration into the host genome, are still unknown. Here, we report that VirF-one of the few known exported Vir proteins whose function in the host cell remains unknown-is involved in targeted proteolysis of VIP1 and VirE2. We show that VirF localizes to the plant cell nucleus and interacts with VIP1, a nuclear protein. VirF, which contains an F-box motif, significantly destabilizes both VIP1 and VirE2 in yeast cells. Destabilization of VIP1 in the presence of VirF was then confirmed in planta. These results suggest that VIP1 and its cognate VirE2 are specifically targeted by the VirF-containing Skp1-Cdc53-cullin-F-box complex for proteolysis. The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of T-DNA expression by a proteasomal inhibitor.
Genetic transformation was first described by Griffith in 1928 and has since been demonstrated in a variety of organisms, including many species of fungi. This review focuses on the history and technology of the transformation of Saccharomyces cerevisiae. The application of protocols developed for S. cerevisiae to other important yeast species is discussed. The protocols for transformation by spheroplasting, LiAc/ssDNA/PEG, and electroporation are compared, and possible mechanisms for transformation are discussed.
Most woody fruit species have long juvenile periods that drastically prolong the time required to analyze mature traits. Evaluation of characteristics related to fruits is a requisite to release any new variety into the market. Because of a decline in regenerative and transformation potential, genetic transformation procedures usually employ juvenile material as the source of plant tissue, therefore resulting in the production of juvenile plants. Direct transformation of mature material could ensure the production of adult transgenic plants, bypassing in this way the juvenile phase. Invigoration of the source adult material, establishment of adequate transformation and regeneration conditions, and acceleration of plant development through grafting allowed us to produce transgenic mature sweet orange trees flowering and bearing fruits in a short time period.
Competence for genetic transformation in Streptococcus pneumoniae is regulated by a quorum-sensing system encoded by two genetic loci, comCDE and comAB. Additional competence-specific operons, cilA, cilB, cilC, cilD, cilE, cinA-recA, coiA, and cfl, involved in the DNA uptake process and recombination, share an unusual consensus sequence at -10 and -25 in the promoter, which is absent from the promoters of comAB and comCDE. This pattern suggests that a factor regulating transcription of these transformation machinery genes but not involved with comCDE and comAB expression might be an alternative sigma factor. A search for such a global transcriptional regulator was begun by purifying pneumococcal RNA polymerase holoenzyme. In preparations from competent pneumococcal cultures a protein which seemed to be responsible for cilA transcription in vitro was identified. The corresponding gene was identified and found to be present in two copies, designated comX1 and comX2, located adjacent to two of the repeated rRNA operons. Expression of transformation machinery operons, such as cilA, cilD, cilE, and cfl, but not that of the quorum-sensing operons comAB and comCDE, was shown to depend on comX, while comX expression depended on ComE but not on ComX itself. We conclude that the factor is a competence-specific global transcription modulator which links quorum-sensing information transduced to ComE to competence and propose that it acts as an alternate sigma factor. We also report that comAB and comCDE are not sufficient for shutoff of competence-stimulating peptide-induced gene expression nor for the subsequent refractory period, suggesting that these phenomena depend on one or more ComX-dependent genes.
Agrobacterium-mediated genetic transformation of plants, a unique example of transkingdom DNA transfer, requires the presence of several proteins encoded by the host cell. One such cellular factor is VIP1, an Arabidopsis protein proposed to interact with and facilitate import of the bacterial DNA-protein transport (T) complexes into the plant cell nucleus. Thus, VIP1 is required for transient expression of the bacterial DNA, an early step in the transformation process. However, the role of VIP1 in subsequent transformation events leading to the stable expression of bacterial DNA was unexplored. Here, we used reverse genetics to dissect VIP1 functionally and demonstrate its involvement in the stable genetic transformation of Arabidopsis plants by Agrobacterium. Our data indicate that the ability of VIP1 to interact with the VirE2 protein component of the T-complex and localize to the cell nucleus is sufficient for transient genetic transformation, whereas its ability to form homomultimers and interact with the host cell H2A histone in planta is required for tumorigenesis and, by implication, stable genetic transformation.
Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.
Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.
Stable red astaxanthin-producing transformants were obtained after genetic transformation of two Phaffia rhodozyma mutants. A yellow mutant, accumulating beta-carotene, and an albino mutant, accumulating phytoene, from P. rhodozyma were transformed using a genomic library of wild-type strain UCD 67-385 in the pBluescript vector. Hybridization assays, using the pBluescript DNA as a radioactive probe, indicate integration of vector sequences into the genome of the transformants. Transformants DNA was digested with restriction endonucleases, ligated with T4 DNA ligase and then used to transform E. coli. Ampicillin resistant plasmids, containing 0.1, 0.2, and 2.5 kb DNA inserts of P. rhodozyma, were rescued from the yeast red transformants. The molecular analysis indicate that transformation has occurred by an integration event of donor DNA into the genome of the host strains.
Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.
Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.