Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single double-strand break (DSB) in a plant genome can be mutagenic due to insertions and deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes, each carrying an I-SceI site. After transient expression of I-SceI a kanamycin-resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by nonhomologous end joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB-induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes.

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It has been proposed that cauliflower mosaic virus 35S RNA with its 600 nt long leader uses an unusual translation process (the translational shunt). A wheat germ in vitro translation assay was used to improve the study of this mechanism. Deletions, the introduction of stable stem-loop structures, and the inhibitory effect of antisense oligonucleotides on gene expression were used to determine the roles of various parts of the leader. It was found that the 5'- and 3'-ends of the leader are absolutely required for translation whereas the middle part is apparently dispensable. These results confirm the data already reported from transient expression experiments with protoplasts. However, the in vitro data suggest in contrast to protoplast experiments that only two relatively short regions at both ends, approximately 100 nt each, are required. The in vitro system provides tools for further studying the shunt model at the molecular level and for examining the involvement of proteins in this mechanism. Shunting was also found to occur with the rice tungro bacilliform virus leader. As wheat is neither a host plant of cauliflower mosaic virus nor rice tungro bacilliform virus, the shunt seems to be host independent, a finding that deviates from earlier studies in protoplasts.

A wheat germ cell-free system was used to study details of ribosome shunting promoted by the cauliflower mosaic virus 35 S RNA leader. By testing a dicistronic construct with the leader placed between two coding regions, we confirmed that the 35 S RNA leader does not include an internal ribosome entry site of the type observed with picornavirus RNAs. A reporter gene fused to the leader was shown to be expressed by ribosomes that had followed the bypass route (shunted) and, with lower efficiency, by ribosomes that had scanned through the whole region. Stem section 1, the most stable of the three stem sections of the leader, was shown to be an important structural element for shunting. Mutations that abolished formation of this stem section drastically reduced reporter gene expression, whereas complementary mutations that restored stem section 1 also restored shunting. A micro-leader capable of shunting consisting of stem section 1 and flanking sequences could be defined. A small open reading frame preceding stem section 1 enhances shunting.

The intergenic spacer (IGS) of the ribosomal DNA spans the region from the 3' end of the 25S rRNA gene to the 5' end of the 18S rRNA gene [12,14].Here we report the sequence of the IGS of tomato (Lycopersicon esculentum) cv. Rutgers. High molecular weight DNA was isolated [ 8 ], the rDNA was enriched in a CsCl-actinomycin D gradient [ 11] and used for restriction analysis and cloning. Unlike in most of the plants investigated [ 12,14] no length heterogeneity could be detected in the tomato rDNA repeating units. A 3.6 kb Eco RI and a 1.0 kb Barn HI fragment, comprising the entire IGS and the flanking coding regions, were ligated to pBR322 vectors and then transferred into competent HB101 cells. After sub-cloning sequence analysis according to Chen and Seeburg [3] was performed in an overlapping manner using dGTP and in addition dITP to avoid band compressions.The entire sequence of the IGS of tomato ( Fig. 1) is 3253 bp long with a G + C content of 53.7~o. The end points of the flanking coding regions were inferred from sequence comparison and are not shown in the figure.Examination of the sequence revealed the presence of two different tandemly arranged repetitive elements denoted RE I and RE II. As indicated in Fig. 1, RE I is present eight times (I/i-I/8) with an average length of 53 bp and a G + C content of 63.6~o. RE II is present six times (II/1-II/6) with an average length of 141 bp and a G + C content of 63.9~o. The two different repeats are separated from each other by an extremely ATrich region (A + T content 66~o). The putative transcription initiation region with the TATA and G G G G G boxes [12,17] is located within this AT-rich region directly in front of the RE II repeats between nt 1563 and nt 1575. At present the IGS sequences of maize [13, 17], rye [1], cucumber [6], radish [4], wheat [2] and mung bean [7] are known. Their comparison shows that they widely diverge in length, nucleotide sequence and organization which has greatly hampered the understanding of the presumed identical regulatory functions of these regions for transcription [ 1,5 ].Tomato is a widely used host plant for various viroids including potato spindle tuber viroid (PSTVd) which accumulates in the nucleoli of infected tomato cells [ 16]. Since the transcription and processing of rRNA takes place in the nucleolus [10] it has been surmised [15] that viroids could exert their pathogenicity via interaction with the IGS. When we compared the PSTVd sequence with the tomato IGS sequence we found many short stretches of limited similarity along the entire IGS chain. However, in all RE II repeats we detected a stretch of 25 nt out of which 19nt are identical with nt60-83 [9] of the The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X14639.

The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (Lycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 bp long with a G + C content of 49.6%. Its sequence exhibits 94%-96% positional identity when it is colinearly aligned with the previously reported sequences of the 17-18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.

Because of the availability of the complete sequence of the genome of the model plant Arabidopsis and of insertion mutants for most genes in public mutant collections, the elucidation of the particular role of different factors involved in DNA recombination and repair processes, an important task for plant biology, is becoming feasible. An assay system based on transgenes harboring homologous overlaps of the beta-glucuronidase (uidA) gene is available to determine recombination behavior in various mutant backgrounds. Restoration of the marker gene by homologous recombination can be detected by histochemical staining in planta. Inclusion of a site of the rare cutting restriction enzyme I-SceI in the transgene construct enables the determination of recombination frequencies after induction of double-strand breaks. In this chapter we describe how the respective transgene is transferred by transformation or crossing into the mutant background, how recombination frequencies are determined, and, if necessary, how cells carrying a restored uidA gene can be isolated and propagated for molecular analysis of the particular recombination event.

UV crosslinking assays were performed to characterize interactions between the leader of the cauliflower mosaic virus (CaMV) 35S RNA and cellular, cytoplasmic proteins. From turnip, a host for CaMV, three different proteins, p35, p49, and p100, interacted with multiple binding sites within the leader p35 binds to RNA nonspecifically and p49 binds with low specificity, whereas p100 interacts specifically with viral sequences. The expression of the proteins is not induced upon virus infection, as there is no difference in the protein pattern between healthy and infected cell extracts. In a cellular fractionation, p35 and p100 remain in the high-speed supernatant whereas p49 cosediments with polysomes. A possible involvement of these proteins in the translation of the CaMV 35S RNA is discussed.