Barbara Hohn

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Wheat mutation rate after Chernobyl

The accident at the Chernobyl nuclear power plant in 1986 has generated concern over the genetic consequences of chronic exposure to radiation. Here we describe a new approach to monitoring germline mutation in plants and find evidence for a remarkably strong induction of germline mutation in wheat upon chronic exposure to ionizing radiation produced by the Chernobyl accident.

Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T0lox plants was crossed with T0 Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T1 plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T1 hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T2 plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

Extremely complex pattern of microsatellite mutation in the germline of wheat exposed to the post-Chernobyl radioactive contamination.

The molecular structure of rare variants at 13 microsatellite loci found in a population of wheat plants grown for one generation in the heavily contaminated 30 km exclusion zone around the Chernobyl Nuclear Power Plant and in a control population was compared. Evidence for rare alterations (variants) was obtained for all 13 loci, including gain and loss of repeats, as well as the complete loss of microsatellite bands. The ratio between gains and losses among variants in the control group was similar to that in the exposed group. Sequencing of variants at six microsatellite loci found in the exposed population revealed extremely complex pattern of germline mutations, including complete deletions of loci, a bias towards mutations with gains and losses of multiple repeat units, and relatively frequent insertions of DNA of unknown origin. The occurrence of large deletions at two loci may be attributed to direct and inverted repeats sequences located just upstream and downstream of the array. The results of our study also suggest that the majority of mutations within the studied wheat microsatellite loci are represented by gains and losses of multiple repeat units, implying that a simple model of replication slippage cannot account for mutation events at these loci. Our data also support the conclusion that the spectra of spontaneous and radiation-induced mutation in wheat may be similar.

Mechanisms of T-DNA integration

T-DNA integration is the final step of the transformation process. During this step, the T-DNA, which traveled as a single-stranded DNA molecule from the bacterial cell through the host-cell cytoplasm into the nucleus, must covalently attach itself to the host to its point of integration in the host genome, to be stripped of some, if not all, of its bacterial and host escorting proteins, and to interact with and co-opt the host’s DNA-repair proteins and machinery for its complementation into a double-stranded DNA molecule during its integration into the host genome. In the following chapter, we describe the current knowledge on the functions performed by the bacterial and host proteins, and the role that the host genome may play, during the integration process. We also present the dominant models used today to explain the complex mechanism of T-DNA integration in plant cells. cell’s double-stranded genomic DNA. To fulfil its destiny, the T-DNA needs to be directed 396 Alicja Ziemienowicz, Tzvi Tzfira and Barbara Hohn

Intrachromosomal Recombination Between Genomic Repeats

Three types of homologous recombination (HR) in plants are reviewed in this book: n n nextrachromosomal recombination (ECR) between input DNAs, either viral replicons (reviewed in chapter 2), or non-replicating input DNAs (chapter 5); n n nrecombination between input (extrachromosomal) DNA and a chromosomally located target DNA sequence, i.e. gene targeting (GT), either of the nuclear genome (chapter 9) or chloroplast genomes (chapter 4); and n n nintrachromosomal recombination (ICR) of the chloroplast (chapter 4), mitochondrial (chapter 3) or nuclear genomes that we review in this chapter.

Agroinfection as a tool for the Investigation of Plant-Pathogen Interactions

The fascinating way by which Agrobacterium invades a host plant has been the subject of intensive investigations by various laboratories, and has opened new avenues of research (reviewed by other authors in this volume, and in references quoted in their articles). Most of these avenues of research utilize the uncanny ability of this bacterium to effect an inter-kingdom type of exchange of genetic information, namely to transfer sequences of nucleic acids from bacterium to plant. This genetic exchange begins with the detection of substances released by wounded plant cells, to which the bacterium reacts by chemotaxis and induction of the bacterial virulence genes necessary for DNA transfer, and ends with the integration and expression of bacterial genes in the plant, that direct production of a plant tumour.

Wheat mutation rate after Chernobyl.

The accident at the Chernobyl nuclear power plant in 1986 has generated concern over the genetic consequences of chronic exposure to radiation. Here we describe a new approach to monitoring germline mutation in plants and find evidence for a remarkably strong induction of germline mutation in wheat upon chronic exposure to ionizing radiation produced by the Chernobyl accident.