When first asked to write a review of my life as a scientist, I doubted anyone would be interested in reading it. In addition, I did not really want to compose my own memorial. However, after discussing the idea with other scientists who have written autobiographies, I realized that it might be fun to dig into my past and to reflect on what has been important for me, my life, my family, my friends and colleagues, and my career. My life and research has taken me from bacteriophage to Agrobacterium tumefaciens–mediated DNA transfer to plants to the plant genome and its environmentally induced changes. I went from being a naïve, young student to a postdoc and married mother of two to the leader of an ever-changing group of fantastic coworkers—a journey made rich by many interesting scientific milestones, fascinating exploration of all corners of the world, and marvelous friendships.
INTRODUCTION This paper is addressed particularly (but not exclusively) to those who use λ as a vector for cloning genes of other organisms. In it we describe techniques for growing and purifying phage and offer a brief menu of tests that are useful for verification of genetic characters found in some widely used vectors. We also suggest methods for simple strain construction, mutagenesis, DNA purification, and in vitro DNA packaging. Alternative and overlapping procedures can also be found (Miller 1972; Davis et al. 1980; Maniatis et al. 1982; Berman et al. 1983). We emphasize that the choice of route is often a matter of individual preference and that methods can and should be varied to suit the particular phage strain, laboratory conditions, and the convenience of the investigator. We have indicated why certain manipulations are to be preferred or avoided, but we warn the reader that the reasons are often speculative and are presented to demystify our subject rather than to inhibit experimentation. GROWTH AND STORAGE OF BACTERIAL HOST STRAINS The commonly used hosts for the growth of phage λ are derivatives of Escherichia coli K12. Those few strains to which particular reference is made in this paper are given in Table 1. All known genes of E. coli K12 and their symbols are listed by Bachmann and Low (1980). For the titration and propagation of λ , bacteria are best grown in a rich medium, such as tryptone broth, with aeration at 37°C, and used when they have reached a...
Genetics allows the elucidation of gene function through the analysis of gene malfunction. Modern genetics and genomics require ways for in situ modification of genes, by means of point mutations, deletions, and additions. The availability of sequence information of many organisms dictates rapid development of reverse genetics procedures. Until recently, targeting of genes with the help of introduced homologous sequences, here referred to as homologous recombination-dependent gene targeting (hrdGT), was the method of choice, at least for mammalian systems. However, in higher eukaryotic organisms such as mammals and plants, exogenously introduced DNA preferably integrates in random positions in the genome, by the process of illegitimate recombination, and only infrequently can targeted integration events be detected. Recently an alternative strategy became available for precise reverse genetics. Specific chimeric oligonucleotides, COs, consisting of DNA and RNA stretches, were found to induce point mutations in several mammalian genes tested (see below). This technique, here referred to as chimeric oligonucleotide-dependent mismatch repair, cdMMR, has now been used for plants: this issue of the Proceedings includes two reports describing stable changes in the genomes of tobacco and maize after treatment with chimeric oligonucleotides (1, 2).
Marker-transgene-dependent lines of Arabidopsis thaliana measuring somatic homologous recombination (SHR) have been available for almost two decades. Here we discuss mechanisms of marker-gene restoration, comment on results obtained using the reporter lines, and stress how caution must be applied to avoid experimental problems or false interpretation in the use of SHR reporter lines. Although theoretically possible, we conclude that explanations other than SHR are unlikely to account for restoration of marker gene expression in the SHR lines when used with appropriate controls. We provide an overview of some of the most important achievements obtained with the SHR lines, give our view of the limitations of the system, and supply the reader with suggestions on the proper handling of the SHR lines. We are convinced that SHR lines are and will remain in the near future a valuable tool to explore the mechanism and influence of external and internal factors on genome stability and DNA repair in plants.
Crude protein extracts of induced and uninduced octopine wild-type strain of Agrobacterium tumefaciens, as well as several mutants of the virulence loci virA, -B, -G, -C, -D, and -E, were probed with single- and double-stranded synthetic oligodeoxynucleotides of different sequence and length in an electrophoretic retardation assay. Four complexes involving sequence-nonspecific, single-stranded-DNA-binding proteins were recognized. One inducible complex is determined by the virE locus, two Ti-plasmid-dependent complexes are constitutively expressed, and a fourth one is controlled by chromosomal genes. The protein-DNA complexes were characterized by sucrose density gradient centrifugation and by determination of the length of single-stranded DNA required for their formation. It is hypothesized that the single-stranded-DNA-binding proteins are involved in the production of T-DNA intermediates or have a carrier or protective function during T-DNA transfer.
