Abstract. The development of functional blood and lymphatic vessels requires spatio-temporal coordination of the production and release of growth factors such as vas-

Although the generation of transgenic plants is now routine, the integration of foreign genetic information has so far been at random sites in the genome. We now present evidence for directed integration into a predicted location in the host plant genome. Protoplasts of transgenic tobacco (Nicotiana tabaccum) plants carrying copies of a partial, non‐functional drug‐resistance gene in the nuclear DNA were used as recipients for DNA molecules containing the missing part of the gene. Molecular and genetic data confirm the integration of the foreign DNA through homologous recombination within overlapping parts of the protein coding region, resulting in the formation of an active gene in the host chromosome. This approach is referred to as gene targeting. The gene targeting frequency (the number of drug‐resistant clones resulting from gene correction compared to the number of resistant clones from parallel experiments with a similar non‐interrupted hybrid gene) was 0.5‐4.2×10‐4. These experiments demonstrate the possibility of producing transgenic plants with desired modifications to a specific nuclear gene.

SummaryPrecise modi®cation by gene targeting (GT) provides an important tool for studies of gene function in vivo. Although routine with many organisms, only isolated examples of GT events have been reported for¯owering plants. These were at low frequencies precluding reliable estimation of targeting ef®ciency and evaluation of GT mechanisms. Here we present an unambiguous and straightforward system for detection of GT events in Arabidopsis using an endogenous nuclear gene encoding protoporphyrinogen oxidase (PPO), involved in chlorophyll and heme syntheses. Inhibition of PPO by the herbicide Butafenacil results in rapid plant death. However, the combination of two particular mutations renders PPO highly resistant to Butafenacil. We exploited this feature for selection of GT events by introducing the mutations into the PPO gene by homologous recombination. We have estimated the basal GT frequency to be 2.4 Q 10 ±3 . Approximately one-third of events were true GT (TGT) leading to the anticipated modi®cation of the chromosomal PPO copy. The remaining events could be classi®ed as ectopic GT (EGT) arising by modi®cation of vector DNA by the chromosomal template and its random integration into the Arabidopsis genome. Thus the TGT frequency in our experimental setup is 0.72 Q 10 ±3 . In view of the high ef®ciency of Arabidopsis transformation, GT experiments of a reasonable size followed by a PCR screen for GT events should also allow for modi®cation of non-selectable targets. Moreover, the system presented here should contribute signi®cantly to future improvement of GT technology in plants.

Levels of induced homologous recombination between chromosomal repeats in plant somatic cells were examined. Transgenic plants of Nicotiana tabacum hemi-or homozygous for pairs of deletion derivatives of the neomycin phosphotransferase (nptI) marker gene integrated at a single genomic locus were produced. Homologous recombination within the overlapping parts of the nptll gene restored the function and the resulting kanamycin resistance was used for scoring recombination frequency. The recombination events were confirmed by the appearance of a characteristic 1245-base-pair EcoRV fragment detected in all kanamycin-resistant clones tested. The rate of spontaneous recombination was found to be related to the copy number of recombination substrates and was 9 x 10-5 and 19 x 10-5 for hemi-and homozygote strains, respectively. Ionizing radiation, mitomycin C, and heat shock markedly increased the frequency of intrachromosomal recombination. Low doses of x-rays (1.25 Gy) enhanced the relative recombination frequency to approximately twice the spontaneous value. The presence of mitomycin C increased the frequency of recombination 9-fold and exposure to an elevated temperature (50°C) increased it 6.5-fold. The x-ray and heat shock treatments reduced cell viability to 53% and 8%, respectively. Mitomycin C treatment had no effect on cell survival.

SummaryThe Arabidopsis MIM gene encodes a protein belonging to the SMC family (structure maintenance of chromosomes) which is required for intrachromosomal homologous recombination (ICR). Both ICR and MIM gene expression are enhanced by DNA-damaging treatments, suggesting that MIM is a factor limiting DNA repair by homologous recombination (HR) under genotoxic stress. We tested this hypothesis by measuring the levels of recombination in the mim mutant under genotoxic stress, using methyl methanesulfonate. Although the mutant clearly showed diminished basal and induced levels of ICR, enhancement of ICR by DNA-damaging treatments was similar to that observed in the wild type. This suggests that the MIM gene product is required for DNA repair by HR, but is not critical for HR induction. To determine whether enhanced availability of MIM would increase basal HR levels in Arabidopsis, we examined ICR frequencies in transgenic Arabidopsis strains overexpressing the MIM gene after ectopic insertion of additional MIM copies. Two independent lines showed a twofold increase in ICR frequency relative to the wild type. Thus MIM is required for ef®cient ICR in plants, and its manipulation can be used to change homologous recombination frequencies. Since MIM is one of the components responsible for chromatin dynamics, our results suggest that the chromatin environment determines the frequency of homologous recombination.