DNA Transfer Process Research Articles

Identification of Relaxase-DNA Covalent Complexes and DNA Strand Transfer Reaction Products by Polyacrylamide Gel Electrophoresis.

Relaxases are essential proteins for plasmid conjugation. They process the DNA to be transferred by means of a covalent intermediate. Thus, the characterization of these covalent complexes is essential to understand the biological role of this reaction and to improve it for biotechnological applications. In this article, we describe the use of the polyacrylamide electrophoresis techniques for the identification of relaxase-DNA covalent complexes, being SDS-PAGE a simple and reliable method for the detection of protein-DNA covalent adducts. Relaxases also perform a strand transfer reaction to recircularize the DNA and finish the DNA transfer process in the recipient cell. Urea-PAGE allows us the analysis of oligonucleotides generated by the strand transfer reaction. These methods could also be used for the analysis of other HUH endonucleases.

Pathways of DNA Transfer to Plants from Agrobacterium tumefaciens and Related Bacterial Species.

Genetic transformation of host plants by Agrobacterium tumefaciens and related species represents a unique model for natural horizontal gene transfer. Almost five decades of studying the molecular interactions between Agrobacterium and its host cells have yielded countless fundamental insights into bacterial and plant biology, even though several steps of the DNA transfer process remain poorly understood. Agrobacterium spp. may utilize different pathways for transferring DNA, which likely reflects the very wide host range of Agrobacterium. Furthermore, closely related bacterial species, such as rhizobia, are able to transfer DNA to host plant cells when they are provided with Agrobacterium DNA transfer machinery and T-DNA. Homologs of Agrobacterium virulence genes are found in many bacterial genomes, but only one non-Agrobacterium bacterial strain, Rhizobium etli CFN42, harbors a complete set of virulence genes and can mediate plant genetic transformation when carrying a T-DNA-containing plasmid.

DNA Delivery and Genomic Integration into Mammalian Target Cells through Type IV A and B Secretion Systems of Human Pathogens.

We explore the potential of bacterial secretion systems as tools for genomic modification of human cells. We previously showed that foreign DNA can be introduced into human cells through the Type IV A secretion system of the human pathogen Bartonella henselae. Moreover, the DNA is delivered covalently attached to the conjugative relaxase TrwC, which promotes its integration into the recipient genome. In this work, we report that this tool can be adapted to other target cells by using different relaxases and secretion systems. The promiscuous relaxase MobA from plasmid RSF1010 can be used to deliver DNA into human cells with higher efficiency than TrwC. MobA also promotes DNA integration, albeit at lower rates than TrwC. Notably, we report that DNA transfer to human cells can also take place through the Type IV secretion system of two intracellular human pathogens, Legionella pneumophila and Coxiella burnetii, which code for a distantly related Dot/Icm Type IV B secretion system. This suggests that DNA transfer could be an intrinsic ability of this family of secretion systems, expanding the range of target human cells. Further analysis of the DNA transfer process showed that recruitment of MobA by Dot/Icm was dependent on the IcmSW chaperone, which may explain the higher DNA transfer rates obtained. Finally, we observed that the presence of MobA negatively affected the intracellular replication of C. burnetii, suggesting an interference with Dot/Icm translocation of virulence factors.

Origin of Nuclear Mitochondrial Pseudogenes (Numts)

Mitochondria are well-defined cytoplasmic organelles of eukaryotic cells, which take part in various cellular metabolic functions, mainly cellular respiration. Several researchers have tried to validate the mechanism of gene transfer between the mitochondria and the cell nucleus. However, some scientific facts show it is very difficult or even impossible for a researcher to confirm DNA transfer process from a living organelle, because mitochondria possess certain specific features that prevent certainty of the DNA transfer process: Such as; dynamicity of mitochondrial organelles, exist in big numbers, can change its location, size and shape, however, gene transfer techniques lack precision. Healthy active mitochondrion has perfect double membranes that sustain its function of keeping in all its constituents. So, most probably the pseudogenes or Numts that are detected in eukaryotic nuclei do not come from living mitochondria rather from aged degraded ones. However, it may come from misplaced DNA segments resulted from any gene transfer process.

Impact of Donor Age, Gender and Handling Time on the DNA Concentration Left on Different Surfaces

We analyzed the correlation between several factors (donor age and gender, and handling time) and trace DNA concentration that participants left on different surfaces (paper, plastic, plastic coated metal) while holding items in their hands or rubbing them with their fingers, their palms, and the side of the palm of the dominant hand. Sixty participants took part in the study. Items were swabbed with a moistened cotton swab. DNA was isolated using the Chelex procedure and quantified by real-time PCR. We found that DNA concentration transferred to an item was independent of the handling time. On the contrary, it was dependent on the item’s texture ; the greatest concentration was left on plastic coated metal and the least on paper. The greatest concentration of trace DNA was left by participants from 35 to 44 years of age. Results of the study showed that men deposit a higher DNA concentration than do women. Item texture, donor age, and gender influence trace DNA concentration. Further investigations are necessary to fully understand the process of DNA transfer from donors to handled items.

UV-inducible DNA exchange in hyperthermophilic archaea mediated by type IV pili

Archaea, like bacteria and eukaryotes, contain proteins involved in various mechanisms of DNA repair, highlighting the importance of these processes for all forms of life. Species of the order Sulfolobales of hyperthermophilic crenarchaeota are equipped with a strongly UV-inducible type IV pilus system that promotes cellular aggregation. Here we demonstrate by fluorescence in situ hybridization that cellular aggregates are formed based on a species-specific recognition process and that UV-induced cellular aggregation mediates chromosomal marker exchange with high frequency. Recombination rates exceeded those of uninduced cultures by up to three orders of magnitude. Knockout strains of Sulfolobus acidocaldarius incapable of pilus production could not self-aggregate, but were partners in mating experiments with wild-type strains indicating that one cellular partner can mediate the DNA transfer. Since pilus knockout strains showed decreased survival upon UV treatment, we conclude that the UV-inducible DNA transfer process and subsequent homologous recombination represents an important mechanism to maintain chromosome integrity in Sulfolobus. It might also contribute substantially to the frequent chromosomal DNA exchange and horizontal gene transfer in these archaea in their natural habitat.

The Actin Cytoskeleton Has an Active Role in the Electrotransfer of Plasmid DNA in Mammalian Cells

Electrotransfer of molecules is a well established technique which finds extensive use for gene transfer and holds great promise for anticancer treatment. Despite its widespread application, the mechanisms governing the entry of DNA into the cell and its intracellular trafficking are not yet known. The aim of this study is to unravel the role of the actin cytoskeleton during gene electrotransfer in cells. We performed single-cell level approaches to observe the organization of the actin cytoskeleton in Chinese hamster ovary (CHO) cells. In addition, we performed experiments at the multiple-cell level to evaluate the efficiency of DNA transfer after alteration of the actin cytoskeleton using the drug latrunculin B. Actin patches colocalizing with the DNA at the plasma membrane were observed with additional characteristics similar to those of the DNA aggregates in terms of time, number, and size. The disruption of the microfilaments reduces the DNA accumulation at the plasma membrane and the gene expression. This is the first direct experimental evidence of the participation of the actin cytoskeleton in DNA electrotransfer.

LpqM, a Mycobacterial Lipoprotein-Metalloproteinase, Is Required for Conjugal DNA Transfer inMycobacterium smegmatis

We have previously described a novel conjugal DNA transfer process that occurs in Mycobacterium smegmatis. To identify donor genes required for transfer, we have performed a transposon mutagenesis screen; we report here that LpqM, a putative lipoprotein-metalloproteinase, is essential for efficient DNA transfer. Bioinformatic analyses predict that LpqM contains a signal peptide necessary for the protein's targeting to the cell envelope and a metal ion binding motif, the likely catalytic site for protease activity. Using targeted mutagenesis, we demonstrate that each of these motifs is necessary for DNA transfer and that LpqM is located in the cell envelope. The requirement for transfer is specific to the donor strain; an lpqM knockout mutant in the recipient is still proficient in transfer assays. The activity of LpqM is conserved among mycobacteria; homologues from both Mycobacterium tuberculosis and Mycobacterium avium can complement lpqM donor mutants, suggesting that the homologues recognize and process similar proteins. Lipoproteins constitute a significant proportion of the mycobacterial cell wall, but despite their abundance, very few have been assigned an activity. We discuss the potential role of LpqM in DNA transfer and the implications of the conservation of LpqM activity in M. tuberculosis.

Reconstructing evolution: Gene transfer from plastids to the nucleus

During evolution, the genomes of eukaryotic cells have undergone major restructuring to meet the new regulatory challenges associated with compartmentalization of the genetic material in the nucleus and the organelles acquired by endosymbiosis (mitochondria and plastids). Restructuring involved the loss of dispensable or redundant genes and the massive translocation of genes from the ancestral organelles to the nucleus. Genomics and bioinformatic data suggest that the process of DNA transfer from organelles to the nucleus still continues, providing raw material for evolutionary tinkering in the nuclear genome. Recent reconstruction of these events in the laboratory has provided a unique tool to observe genome evolution in real time and to study the molecular mechanisms by which plastid genes are converted into functional nuclear genes. Here, we summarize current knowledge about plastid-to-nuclear gene transfer in the context of genome evolution and discuss new insights gained from experiments that recapitulate endosymbiotic gene transfer in the laboratory.

Plasmid DNA transfer in Mycobacterium smegmatis involves novel DNA rearrangements in the recipient, which can be exploited for molecular genetic studies.

The establishment of molecular genetic techniques is essential for development of new treatments for mycobacterial infections. To this end, we recently described a novel DNA transfer process that occurs in the model mycobacterial organism Mycobacterium smegmatis. This transfer system is most like conjugal DNA transfer in that it requires two viable parents, is DNAse resistant and occurs between distinct donor and recipient strains. Cis-acting sequences called bom, which confer transferability, are distinct from the prototypical oriT sites of conjugative plasmids, as they occur at multiple locations in the chromosome and require RecA in the recipient to mediate plasmid recircularization. Here, we show that a plasmid containing two of these bom regions can undergo several fates in the recipient cell, each of which require recipient recombination functions. The products of plasmid transfer that we observed provide further insights toward a model for DNA transfer. Furthermore, we have taken advantage of the recombination events that occur in the recipient to develop simple procedures for capturing, or replacing specific segments of the recipient chromosome. To demonstrate the potential of the system, we describe the capture and deletion of 25 kb of the M. smegmatis chromosome, and targeted-allele exchange of the recipient recB and recD genes. Using these transfer-mediated rearrangements, we demonstrate that homology with the recipient chromosome and RecB, but not RecD, are essential for DNA transfer.

The bases of crown gall tumorigenesis.

The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).

DNA transfer from Agrobacterium to plant cells in crown gall tumor disease.

Agrobacterium tumefaciens, a Gram-negative soil bacterium, causes crown gall tumor disease on most plants (Hooykaas and Beijersbergen, 1994; Zambryski, 1992; Kado, 1991; Ream, 1989; Nester et al., 1984). The bacterium infects plants at a wound site and forms tumors at the site of infection. Virulent strains of Agrobacterium contain a large tumor-inducing (Ti-) plasmid. Upon infection the bacterium donates a segment of the Ti-plasmid DNA, the transferred (T-) DNA, to the plant cell, where it is stably incorporated into the nuclear genome. Encoded within the T-DNA are genes for the biosynthesis of plant hormones auxin and cytokinin. Constitutive expression of these genes in a transformed plant results in an uncontrolled growth of cells, a cancerous phenotype. The related bacterium Agrobacterium rhizogenes causes the hairy-root disease that results from the stable transfer of the root-inducing (Ri-) plasmid DNA to plants. The process of DNA transfer from Agrobacterium to plants represents a naturally occurring gene transfer across a phylogenetic barrier.KeywordsAgrobacterium TumefaciensBorder SequenceTransport PoreVirD2 ProteinPlant Cell NucleusThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Transcription of ColE1Ap mbeC induced by conjugative plasmids from twelve different incompatibility groups.

Although nonconjugative mobilizable plasmids require helping functions of conjugative plasmids in order to be mobilized into recipients, at least some genes from the nonconjugative plasmids may be induced to assist in the DNA transfer process. Conjugative plasmids from 12 different incompatibility groups mobilized the nonconjugative plasmid ColE1Ap between Escherichia coli strains. Introduction of any of the conjugative plasmids into the ColE1Ap-containing strain resulted in an induction of mbeC, the product of which is a component of the mobilization relaxation complex. Each of the conjugative plasmids caused protein to bind specifically to mbe promoter DNA, suggesting a direct regulatory interaction.

Long-range structure of H-ras 1-selected transgenomes

We have used chromosome-mediated gene transfer (CMGT) and whole cell fusion to derive human-mouse hybrid cells carrying reduced human chromosomes 11, by selecting for expression of the transforming H-ras 1 oncogene. To realize the full potential of these somatic cell genetic techniques as resources for enriched DNA probe isolation and the fine structure mapping of chromosomes, the nature of any molecular rearrangements that may accompany the process of DNA transfer must be understood. We have analyzed the long-range structure of our transgenomes by pulsed field gel electrophoresis (PFGE) and show here that, whereas during cell fusion several megabase pairs (Mb) of DNA can be transferred intact, multiple rearrangements of DNA accompany CMGT even in transgenomes where other methods of analysis gave no indication of such molecular scrambling.

The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants

Crown gall and hairy root formation in plants involve the transfer of a DNA segment (T-DNA) from Agrobacterium to plant cells. The T-DNA, along with the vir genes which are essential for the DNA transfer process, is carried on a large tumour-inducing (Ti) plasmid in Agrobacterium tumefaciens (see refs 1 and 2 for review). The transfer of T-DNA, the only known case of naturally occurring DNA transfer from bacteria to plants, has been extensively exploited for the genetic transformation of plants3. Conjugal transfer of DNA between bacteria involves the transfer (tra) and mobilization (mob) gene products. The mob proteins nick DNA at the origin of transfer (oriT) and initiate single-stranded transfer of the DNA into the recipient bacterium (see ref. 4 for review). We report here that the mobilization functions on the naturally occurring wide-host-range plasmid RSF1010 can mediate the transfer of plasmids from Agrobacterium into plant cells as well as transfer between bacteria. This suggests that plants have access to the gene pool of Gram-negative bacteria.

Genetic analysis of integration mediated by single T-DNA borders.

Transformation of plant cells by the T-DNA of the Ti plasmid of Agrobacterium tumefaciens depends in part upon a sequence adjacent to the right T-DNA end. When this sequence is absent, the T-DNA is almost avirulent; when it is present, DNA between it and the left T-DNA border region becomes integrated in plants. To investigate further this process of DNA transfer and integration, we introduced the right border region and the nopaline synthase (nos) gene of plasmid pTiC58 into a variety of new positions around Ti plasmids. The border region functioned when separated from the remainder of the T-DNA by almost 50 kilobases. It also worked when placed outside of the T-DNA region where there were no known left-border sequences with which to interact. Indeed, the nos gene could be transferred to plants even when no other Ti plasmid sequences were present on the same plasmid. These results may indicate that the sequence requirements for the left borders are not as stringent as those for the right borders. In addition, mutants with an extra copy of the right border region within their T-DNA were found to transfer or integrate only parts of the bacterial T-DNA region. It is possible that abnormally placed T-DNA borders interfere with the normal process of DNA transfer, integration, or both.

Expression of T6 sensitivity in Escherichia coli K12F minus bacteria after mating with HFR cells.

We have observed that in a mating between phage T6 sensitive (tsx S ) Escherichia coli K12 Hfr and phage T6 resistant F- bacteria, the tsx s allele is expressed in all the zygotes which receive this gene. We have demonstrated that this acquisition of T6 sensitivity reflects the capacity of tsx s to be expressed after the process of DNA transfer independently of the integration of this marker into the F- chromosomes. This expression does not require cell division. Yet, the zygotes which have received, but have not integrated, the tsxs gene yield a progeny which have lost the Tsxs phenotype. The kinetics of the loss of the Txs phenotype is compatible with the hypothesis that tsxs is expressed only during a transient period in the zygotes if it is not integrated into a chromosome. The results obtained are also consistent with the idea that the product of tsxs is a structural protein of the T6 phage receptor.