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Keith M Derbyshire - One of the best experts on this subject based on the ideXlab platform.

  • the specialized secretory apparatus esx 1 is essential for DNA Transfer in mycobacterium smegmatis
    Molecular Microbiology, 2008
    Co-Authors: Abbie Coros, Brian Callahan, Eric Battaglioli, Keith M Derbyshire
    Abstract:

    Conjugal DNA Transfer in Mycobacterium smegmatis occurs by a mechanism distinct from plasmid-mediated DNA Transfer. Previously, we had shown that the secretory apparatus, ESX-1, negatively regulated DNA Transfer from the donor strain; ESX-1 donor mutants are hyper-conjugative. Here, we describe a genome-wide transposon mutagenesis screen to isolate recipient mutants. Surprisingly, we find that a majority of insertions map within the esx-1 locus, which encodes the secretory apparatus. Thus, in contrast to its role in donor function, ESX-1 is essential for recipient function; recipient ESX-1 mutants are hypo-conjugative. In addition to esx-1 genes, our screen identifies novel non-esx-1 loci in the M. smegmatis genome that are required for both DNA Transfer and ESX-1 activity. DNA Transfer therefore provides a simple molecular genetic assay to characterize ESX-1, which, in Mycobacterium tuberculosis, is necessary for full virulence. These findings reinforce the functional intertwining of DNA Transfer and ESX-1 secretion, first described in the M. smegmatis donor. Moreover, our observation that ESX-1 has such diametrically opposed effects on Transfer in the donor and recipient, forces us to consider how proteins secreted by the ESX-1 apparatus can function so as to modulate two seemingly disparate processes, M. smegmatis DNA Transfer and M. tuberculosis virulence.

  • the rd1 virulence locus of mycobacterium tuberculosis regulates DNA Transfer in mycobacterium smegmatis
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Jessica L Flint, Joseph C Kowalski, Pavan K Karnati, Keith M Derbyshire
    Abstract:

    Conjugal DNA Transfer occurs by an atypical mechanism in Mycobacterium smegmatis. The Transfer system is chromosomally encoded and requires recipient recombination functions for both chromosome and plasmid Transfer. Cis-acting sequences have been identified that confer mobility on nonTransferable plasmids, but these are larger and have different properties to canonical oriT sites found in bacterial plasmids. To identify trans-acting factors required for mediating DNA Transfer, a library of transposon insertion mutants was generated in the donor strain, and individual mutants were screened for their effect on Transfer. From this screen, a collection of insertion mutants was isolated that increased conjugation frequencies relative to wild type. Remarkably, the mutations map to a 25-kb region of the M. smegmatis chromosome that is syntenous with the RD1 region of Mycobacterium tuberculosis, which is considered to be the primary attenuating deletion in the related vaccine strain Mycobacterium bovis bacillus Calmette–Guerin. The genes of the RD1 region encode a secretory apparatus responsible for exporting Cfp10- and Esat-6, both potent antigens and virulence factors. In crosses using two M. smegmatis donors, we show that wild-type cells can suppress the elevated Transfer phenotype of mutant donors, which is consistent with the secretion of a factor that suppresses conjugation. Most importantly, the RD1 region of M. tuberculosis complements the conjugation phenotype of the RD1 mutants in M. smegmatis. Our results indicate that the M. tuberculosis and M. smegmatis RD1 regions are functionally equivalent and provide a unique perspective on the role of this critical secretion apparatus.

Roland A.h. Van Oorschot - One of the best experts on this subject based on the ideXlab platform.

  • DNA Transfer in forensic science a review
    Forensic Science International-genetics, 2019
    Co-Authors: Roland A.h. Van Oorschot, Bianca Szkuta, Georgina E Meakin, Bas Kokshoorn, Mariya Goray
    Abstract:

    Abstract Understanding the variables impacting DNA Transfer, persistence, prevalence and recovery (DNA-TPPR) has become increasingly relevant in investigations of criminal activities to provide opinion on how the DNA of a person of interest became present within the sample collected. This review considers our current knowledge regarding DNA-TPPR to assist casework investigations of criminal activities. There is a growing amount of information available on DNA-TPPR to inform the relative probabilities of the evidence given alternative scenarios relating to the presence or absence of DNA from a specific person in a collected sample of interest. This information should be used where relevant. However, far more research is still required to better understand the variables impacting DNA-TPPR and to generate more accurate probability estimates of generating particular types of profiles in more casework relevant situations. This review explores means of achieving this. It also notes the need for all those interacting with an item of interest to have an awareness of DNA Transfer possibilities post criminal activity, to limit the risk of contamination or loss of DNA. Appropriately trained forensic practitioners are best placed to provide opinion and guidance on the interpretation of profiles at the activity level. However, those requested to provide expert opinion on DNA-related activity level issues are often insufficiently trained to do so. We advocate recognition of DNA activity associated expertise to be distinct from expertise associated with the identification of individuals. This is to be supported by dedicated training, competency testing, authorisation, and regular fit for purpose proficiency testing. The possibilities for experts to report on activity-related issues will increase as our knowledge increases through further research, access to relevant data is enhanced, and tools to assist interpretations are better exploited. Improvement opportunities will be achieved sooner, if more laboratories and agencies accept the need to invest in these aspects as well as the training of practitioners.

  • DNA Transfer by examination tools a risk for forensic casework
    Forensic Science International-genetics, 2015
    Co-Authors: Michelle L. Harvey, Bianca Szkuta, Kaye N. Ballantyne, Roland A.h. Van Oorschot
    Abstract:

    Abstract The introduction of profiling systems with increased sensitivity has led to a concurrent increase in the risk of detecting contaminating DNA in forensic casework. To evaluate the contamination risk of tools used during exhibit examination we have assessed the occurrence and level of DNA Transferred between mock casework exhibits, comprised of cotton or glass substrates, and high-risk vectors (scissors, forceps, and gloves). The subsequent impact of such Transfer in the profiling of a target sample was also investigated. Dried blood or touch DNA, deposited on the primary substrate, was Transferred via the vector to the secondary substrate, which was either DNA-free or contained a target sample (dried blood or touch DNA). Pairwise combinations of both heavy and light contact were applied by each vector in order to simulate various levels of contamination. The Transfer of dried blood to DNA-free cotton was observed for all vectors and Transfer scenarios, with Transfer substantially lower when glass was the substrate. Overall touch DNA Transferred less efficiently, with significantly lower Transfer rates than blood when Transferred to DNA-free cotton; the greatest Transfer of touch DNA occurred between cotton and glass substrates. In the presence of a target sample, the detectability of Transferred DNA decreased due to the presence of background DNA. Transfer had no impact on the detectability of the target profile, however, in casework scenarios where the suspect profiles are not known, profile interpretation becomes complicated by the addition of contaminating alleles and the probative value of the evidence may be affected. The results of this study reiterate the need for examiners to adhere to stringent laboratory cleaning protocols, particularly in the interest of contamination minimisation, and to reduce the handling of items to prevent intra-item Transfer.

  • the complexities of DNA Transfer during a social setting
    Legal Medicine, 2015
    Co-Authors: Mariya Goray, Roland A.h. Van Oorschot
    Abstract:

    When questions relating to how a touch DNA sample from a specific individual got to where it was sampled from, one has limited data available to provide an assessment on the likelihood of specific Transfer events within a proposed scenario. This data is mainly related to the impact of some key variables affecting Transfer that are derived from structured experiments. Here we consider the effects of unstructured social interactions on the Transfer of touch DNA. Unscripted social exchanges of three individuals having a drink together while sitting at a table were video recorded and DNA samples were collected and profiled from all relevant items touched during each sitting. Attempts were made to analyze when and how DNA was Transferred from one object to another. The analyses demonstrate that simple minor everyday interactions involving only a few items in some instances lead to detectable DNA being Transferred among individuals and objects without them having contacted each other through secondary and further Transfer. Transfer was also observed to be bi-directional. Furthermore, DNA of unknown source on hands or objects can be Transferred and interfere with the interpretation of profiles generated from targeted touched surfaces. This study provides further insight into the Transfer of DNA that may be useful when considering the likelihood of alternate scenarios of how a DNA sample got to where it was found.

  • the influence of substrate on DNA Transfer and extraction efficiency
    Forensic Science International-genetics, 2013
    Co-Authors: Timothy J Verdon, John R Mitchell, Roland A.h. Van Oorschot
    Abstract:

    Abstract The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary Transfer. In order to improve the currently problematic evaluation of Transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA Transfer involving blood. DNA Transfer percentages were significantly higher ( p =0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on Transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant ( p =0.03) effect on DNA Transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, Transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant ( p =0.03) differences were found among the extraction efficiencies from different materials. When 15μL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant ( p p

  • investigation of secondary DNA Transfer of skin cells under controlled test conditions
    Legal Medicine, 2010
    Co-Authors: John R Mitchell, Mariya Goray, Roland A.h. Van Oorschot
    Abstract:

    Abstract There is a paucity of data on the relative Transfer rates of deposited biological substances which could assist evaluation of the probability of given crime scene scenarios, especially for those relating to objects originally touched by hand. This investigation examines factors that may influence the secondary Transfer of DNA from this source, including the freshness of the deposit, the nature of the primary and secondary substrate and the manner of contact between the surfaces. The Transfer rates showed that both the primary and secondary type of substrate and the manner of contact are important factors influencing Transfer of skin cells, but, unlike other biological fluids, such as blood and saliva, the freshness of the deposit in most instances is not. Skin cells deposited on a non-porous primary substrate Transferred more readily to subsequent substrates than those deposited on a porous substrate. Porous secondary substrates, however, facilitated Transfer more readily than non-porous secondary substrates, from both porous and non-porous surfaces. Friction as the manner of contact significantly increased the rate of Transfer. The findings of this study improve our general understanding of the Transfer of DNA material contained in fingerprints that is left on a surface, and assist in the evaluation of the probability of secondary and further DNA Transfer under specific conditions.

Eugene W. Nester - One of the best experts on this subject based on the ideXlab platform.

  • agrobacterium tumefaciens mediated transformation of yeast
    Proceedings of the National Academy of Sciences of the United States of America, 1996
    Co-Authors: Kevin L Piers, Joe Don Heath, Xiaoyou Liang, Kathryn M Stephens, Eugene W. Nester
    Abstract:

    Agrobacterium tumefaciens Transfers a piece of its Ti plasmid DNA (Transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can Transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA Transfer into S. cerevisiae is very similar to T-DNA Transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes of A. tumefaciens that are required for T-DNA Transfer into plants are also required for T-DNA Transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA Transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.

  • an essential virulence protein of agrobacterium tumefaciens virb4 requires an intact mononucleotide binding domain to function in Transfer of t DNA
    Molecular Genetics and Genomics, 1994
    Co-Authors: Karla Jean Fullner, Kathryn M Stephens, Eugene W. Nester
    Abstract:

    The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the Transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the Transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA Transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ Transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA Transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid Transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA Transfer.

  • vira the plant signal receptor is responsible for the ti plasmid specific Transfer of DNA to maize by agrobacterium
    Proceedings of the National Academy of Sciences of the United States of America, 1993
    Co-Authors: Deanna M Raineri, M I Boulton, J W Davies, Eugene W. Nester
    Abstract:

    Abstract Agrobacteria exhibit marked Ti (tumor-inducing)/Ri (root-inducing) plasmid specificity in their interaction with the Gramineae. In this study, we have used the technique of "agroinfection," in which Agrobacterium-mediated delivery of viral genomes into plants is detected by the development of viral disease symptoms, to identify the region of the Ti plasmid which is responsible for the major differences seen in the ability of nopaline- vs. octopine-type Ti plasmids to Transfer maize streak virus (MSV) DNA to maize. Introduction of fragments of the C58 (nopaline-type) Ti plasmid into strains containing an octopine-type Ti plasmid showed that a fragment containing the nopaline-type virA locus was able to complement these normally non-agroinfectious strains to high levels of MSV DNA Transfer. Octopine-type virA mutant strains that express vir genes at high levels in the absence of the plant inducing compound acetosyringone also efficiently Transferred MSV DNA. These findings imply a functional difference between the virA gene products encoded by octopine- and nopaline-type Ti plasmids which has a profound effect on their ability to mediate DNA Transfer to maize.

  • genetic analysis of the vird operon of agrobacterium tumefaciens a search for functions involved in transport of t DNA into the plant cell nucleus and in t DNA integration
    Journal of Bacteriology, 1993
    Co-Authors: Zdena Koukolikovanicola, Eugene W. Nester, Bruno Tinland, Deanna M Raineri, K Stephens, C Ramos, Barbara Hohn
    Abstract:

    The Transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species. It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration. Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA Transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration. However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA Transfer. virD4 mutants were shown by agroinfection to be completely inactive in T-DNA Transfer.

Mariya Goray - One of the best experts on this subject based on the ideXlab platform.

  • DNA Transfer in forensic science a review
    Forensic Science International-genetics, 2019
    Co-Authors: Roland A.h. Van Oorschot, Bianca Szkuta, Georgina E Meakin, Bas Kokshoorn, Mariya Goray
    Abstract:

    Abstract Understanding the variables impacting DNA Transfer, persistence, prevalence and recovery (DNA-TPPR) has become increasingly relevant in investigations of criminal activities to provide opinion on how the DNA of a person of interest became present within the sample collected. This review considers our current knowledge regarding DNA-TPPR to assist casework investigations of criminal activities. There is a growing amount of information available on DNA-TPPR to inform the relative probabilities of the evidence given alternative scenarios relating to the presence or absence of DNA from a specific person in a collected sample of interest. This information should be used where relevant. However, far more research is still required to better understand the variables impacting DNA-TPPR and to generate more accurate probability estimates of generating particular types of profiles in more casework relevant situations. This review explores means of achieving this. It also notes the need for all those interacting with an item of interest to have an awareness of DNA Transfer possibilities post criminal activity, to limit the risk of contamination or loss of DNA. Appropriately trained forensic practitioners are best placed to provide opinion and guidance on the interpretation of profiles at the activity level. However, those requested to provide expert opinion on DNA-related activity level issues are often insufficiently trained to do so. We advocate recognition of DNA activity associated expertise to be distinct from expertise associated with the identification of individuals. This is to be supported by dedicated training, competency testing, authorisation, and regular fit for purpose proficiency testing. The possibilities for experts to report on activity-related issues will increase as our knowledge increases through further research, access to relevant data is enhanced, and tools to assist interpretations are better exploited. Improvement opportunities will be achieved sooner, if more laboratories and agencies accept the need to invest in these aspects as well as the training of practitioners.

  • the complexities of DNA Transfer during a social setting
    Legal Medicine, 2015
    Co-Authors: Mariya Goray, Roland A.h. Van Oorschot
    Abstract:

    When questions relating to how a touch DNA sample from a specific individual got to where it was sampled from, one has limited data available to provide an assessment on the likelihood of specific Transfer events within a proposed scenario. This data is mainly related to the impact of some key variables affecting Transfer that are derived from structured experiments. Here we consider the effects of unstructured social interactions on the Transfer of touch DNA. Unscripted social exchanges of three individuals having a drink together while sitting at a table were video recorded and DNA samples were collected and profiled from all relevant items touched during each sitting. Attempts were made to analyze when and how DNA was Transferred from one object to another. The analyses demonstrate that simple minor everyday interactions involving only a few items in some instances lead to detectable DNA being Transferred among individuals and objects without them having contacted each other through secondary and further Transfer. Transfer was also observed to be bi-directional. Furthermore, DNA of unknown source on hands or objects can be Transferred and interfere with the interpretation of profiles generated from targeted touched surfaces. This study provides further insight into the Transfer of DNA that may be useful when considering the likelihood of alternate scenarios of how a DNA sample got to where it was found.

  • DNA Transfer within forensic exhibit packaging potential for DNA loss and relocation
    Forensic Science International-genetics, 2012
    Co-Authors: Mariya Goray, Roland A H Van Oorschot, John Mitchell
    Abstract:

    Crime scene samples after their collection are packaged and transported to the laboratory for examination and DNA analysis. The amount and location of DNA-containing material retrieved from an exhibit can be critical in acquiring a profile for incrimination or exclusion purposes and for elucidating criminal events. This paper shows that significant quantities of DNA are frequently: (a) Transferred from the exhibit to the inside of the packaging and (b) Transferred from its area of initial deposit to other areas of the same exhibit and/or to other exhibits within the same package. There is a distinct possibility of failing to generate adequate profiles in instances where the DNA content may otherwise have been adequate, and for the misinterpretation of a result that could impact negatively on the criminal investigation and court outcome. These findings highlight the need for improvements in the collection and packaging of forensic casework exhibits for DNA analysis.

  • investigation of secondary DNA Transfer of skin cells under controlled test conditions
    Legal Medicine, 2010
    Co-Authors: John R Mitchell, Mariya Goray, Roland A.h. Van Oorschot
    Abstract:

    Abstract There is a paucity of data on the relative Transfer rates of deposited biological substances which could assist evaluation of the probability of given crime scene scenarios, especially for those relating to objects originally touched by hand. This investigation examines factors that may influence the secondary Transfer of DNA from this source, including the freshness of the deposit, the nature of the primary and secondary substrate and the manner of contact between the surfaces. The Transfer rates showed that both the primary and secondary type of substrate and the manner of contact are important factors influencing Transfer of skin cells, but, unlike other biological fluids, such as blood and saliva, the freshness of the deposit in most instances is not. Skin cells deposited on a non-porous primary substrate Transferred more readily to subsequent substrates than those deposited on a porous substrate. Porous secondary substrates, however, facilitated Transfer more readily than non-porous secondary substrates, from both porous and non-porous surfaces. Friction as the manner of contact significantly increased the rate of Transfer. The findings of this study improve our general understanding of the Transfer of DNA material contained in fingerprints that is left on a surface, and assist in the evaluation of the probability of secondary and further DNA Transfer under specific conditions.

  • secondary DNA Transfer of biological substances under varying test conditions
    Forensic Science International-genetics, 2010
    Co-Authors: Ecec E Eken, Mariya Goray, R J Mitchell, Roland A.h. Van Oorschot
    Abstract:

    This research investigates factors that may influence the secondary Transfer of DNA. These include the type of biological substance deposited, the nature of the primary and secondary substrate, moisture content of the deposit and type of contact between the surfaces. Results showed that secondary Transfer is significantly affected by both the type of primary substrate and the moisture (wetness) of the biological sample. Porous substrates and/or dry samples diminished Transfer (with on average only 0.36% of biological material being Transferred from one site to another), whereas non-porous substrates and/or wet samples facilitated Transfer events (approximately 50–95% of biological material was Transferred from one site to another). Further, the type of secondary substrate also influenced Transfer rate, with porous surfaces, absorbing Transferred biological substances more readily than non-porous ones. No significant differences were observed among the biological substances tested (pure DNA, blood and saliva). Friction contact between the two substrates significantly enhanced secondary Transfer compared to either passive or pressure contact. These preliminary results will assist in developing general assumptions when estimating probability of a secondary DNA Transfer event under simple conditions.

Paul J J Hooykaas - One of the best experts on this subject based on the ideXlab platform.

  • t DNA from agrobacterium tumefaciens as an efficient tool for gene targeting in kluyveromyces lactis
    Molecular Genetics and Genomics, 1999
    Co-Authors: Paul Bundock, K Mroczek, Aaron Adriaan Winkler, H Y Steensma, Paul J J Hooykaas
    Abstract:

    The soil bacterium Agrobacterium tumefaciens can Transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to Transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate Transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA Transfer (electroporation) or T-DNA Transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used.

  • trans kingdom t DNA Transfer from agrobacterium tumefaciens to saccharomyces cerevisiae
    The EMBO Journal, 1995
    Co-Authors: Paul Bundock, Den A Dulkras, Alice Beijersbergen, Paul J J Hooykaas
    Abstract:

    Abstract Agrobacterium tumefaciens Transfers part of its tumour-inducing (Ti) plasmid, the Transferred or T-DNA, to plants during tumourigenesis. This represents the only example of naturally occurring trans-kingdom Transfer of genetic material. Here we report that A.tumefaciens can Transfer its T-DNA not only to plant cells, but also to another eukaryote, namely the yeast Saccharomyces cerevisiae. The Ti plasmid virulence (vir) genes that mediate T-DNA Transfer to plants were found to be essential for Transfer to yeast as well. Transgenic S.cerevisiae strains were analysed for their T-DNA content. Results showed that T-DNA circles were formed in yeast with precise fusions between the left and right borders. Such T-DNA circles were stably maintained by the yeast if the replicator from the yeast 2 mu plasmid was present in the T-DNA. Integration of T-DNA in the S.cerevisiae genome was found to occur via homologous recombination. This contrasts with integration in the plant genome, where T-DNA integrates preferentially via illegitimate recombination. Our results thus suggest that the process of T-DNA integration is predominantly determined by host factors.

  • factors affecting the rate of t DNA Transfer from agrobacterium tumefaciens to nicotiana glauca plant cells
    Plant Molecular Biology, 1992
    Co-Authors: Teresa Mozo, Paul J J Hooykaas
    Abstract:

    Different factors involved in the early steps of the T-DNA Transfer process were studied by using a β-glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA Transfer was shown to be stimulated in the case of non-oncogenic strains by the co-Transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA Transfer rate especially in virC mutants. The virC mutants Transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.