Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1–7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5–10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.     

No More Foreigners? The Remaking of German Naturalization and Citizenship Law, 1990–2000

Since the beginning of the 1990s, Germany has been taking controversial policy measures that had been quite unimaginable, even in the 1980s. On the one hand, regulations for asylum seekers and ethnic Germans have gradually become restrictive in nature. On the other hand, changes to the Citizenship and Nationality Law of 1913 (Reichs und Staatsangehörigkeitsgesetz) and the Alien Act of 1965 (Ausländergesetz) have dramatically altered the naturalization process. Before the passage of the new German citizenship law in 1999, the acquisition of citizenship in Germany was solely based on the principle of descent (jus sanguinis). This paper attempts to explain why German government altered its citizenship policy and introduced birthright citizenship. It reviews the post-war contextual factors that have shaped the debate on citizenship and asks whether it is external or internal factors, that is international-level norms and institutions or domestic politics, that led to the change. If domestic politics can fully explain the adoption of the new citizenship law, what domestic forces played an important role? Was state action taken in the name of protecting “the national interest” or was it party politics? What role did societal forces play in the making of new citizenship policy? Did the act seek to mitigate anti-foreigner sentiment growing in Germany since the 1990s? Using archival and documentary research and secondary data on socio-economic and demographic trends in Germany, this paper concludes that domestic politics explain the changes in the citizenship law. I argue that granting and withholding national citizenship, including birthright citizenship, is exclusively in the hands of a nation state. Considering a purely ethnocultural understanding of nation-hood now includes legal and territorial components, the new German citizenship law is a major reform at the both theoretical and conceptual level. However, the practical consequences of the new naturalization process remain to be seen. The main question for the future research agenda is whether the new naturalization and citizenship laws will bring full integration of foreigners living in German society.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Functional analysis of the Agrobacterium tumefaciens octopine Ti-plasmid left and right T-region border fragments

Summary Border fragments of the octopine Ti-plasmid were tested for their ability to restore tumorigenicity of an avirulent mutant carrying a deleted right border. It was found that neither introduction of left border fragments nor that of small right border fragments at the position of the deletion resulted in a complete restoration of oncogenicity. However, insertion of a larger right border fragment in the deletion mutant gave fully oncogenic strains. In the latter case sequences to the right side of the right border repeat were found to be responsible for a complete restoration of oncogenicity. Also a left border repeat inserted together with this enhancer sequence fully restored the oncogenicity of the deletion mutant. The enhancer-sequence on itself was not able to mediate the transfer of the T-region to the plant cell. Border fragments inserted in inverted orientation in the deletion mutant were able to mediate the transfer of the T-region to the plant cell, but at a reduced frequency.     

Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

Structure of T-DNA in plants regenerated from roots transformed by Agrobacterium rhizogenes strain A4

Summary The structure of the T-DNA in Ri-transformed plants of Brassica napus, Nicotiana plumbaginifolia and Nicotiana tabacum was analysed. All the plants studied present a particular phenotype with wrinkled leaves. The T-DNA is composed of two parts: TL and TR. The size of the TL-DNA (19–20 kb) seems to be almost constant, except in N. tabacum where it is shorter. The TR-DNA can be absent, and its size varies from about 5–28 kb, with two predominant lengths. The smaller size does not include the region homologous to the tms genes of the pTi T-DNA. The copy number varies from one to four copies per plant genome. TL and TR-DNA are not always present in the same copy number, but in some cases are linked together.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Physiological integration in Cassia fasciculata Michx.: inflorescence removal and defoliation experiments

Summary In the annual herb Cassia fasciculata virtually every leaf subtends an axillary inflorescence. We examined the degree to which these leaf-inflorescence units (reproductive nodes) were physiologically independent of each other in the production of flowers, fruits, and seeds. Removal of up to 4 of every 5 inflorescences resulted in substantial increases in fruit and seed production by remaining, intact reproductive nodes. These increases nearly compensated for and manipulated reproductive nodes were associated with different vascular strands. When 2 of every 3 leaves were removed, fruit and seed production were reduced at both intact and defoliated reproductive nodes. Taken together, these results suggest that neighboring reproductive nodes in C. fasciculata are not physiologically independent of one another, and that competition among fruits and seeds for parental resources occurs over several reproductive nodes.Scientific contribution no. 1595 from the New Hampshire Agricultural Experiment Station