Ac-induced homologous recombination in transgenic tobacco
--Shalev, G and Levy, AA

Ac has been shown to induce intrachromosomal recombination between direct repeats flanking Ac insertion in the maize P locus (Athma and Peterson, Genetics 128:163-173). We have further investigated Ac-induced homologous recombination (HR) in transgenic tobacco plants transformed with the constructs described in Figure 1. Our recombination assay is based on reactivation of the ß-Glucuronidase (GUS) gene following ectopic HR between two defective GUS genes. In this assay, one HR partner carries the pGS001 construct (3' deleted GUS gene). The second HR partner carries either the pGS008 construct (5' GUS deletion and Ac between the 35S promoter and the deletion), or pGS009 (5' GUS deletion and Ds between the 35S promoter and the deletion). T1 plants transformed with pGS001 were crossed with T1 plants transformed with pGS008 or pGS009. Blue sectors, following X-Gluc in-situ staining of F1 seedlings, were detected only in crosses with pGS008, i.e. in the presence of Ac but not Ds. These events are interpreted as Ac-induced somatic recombination between ectopic sequences. Data summarized in Table 1 suggest that Ac enhances ectopic recombination by at least two orders of magnitude. We are in the process of physically characterizing these putative recombination events.

Table 1. Frequency and localization of blue sectors in seedlings.
 
Seedling population
No. of stained seedlingsa
No. of blue sectors detected in various seedling organsb
   
R
H
C
1st
Total
F1 (5'DGUS:Ac X 3'DGUS)
1266
46
53
50
24
173
F1 (5'DGUS:Ds X 3'DGUS)
2400
0
0
0
0
0
3'DGUS
3300
0
0
0
0
0
5'DGUS:Ac
1500
0
0
0
0
0
wild type tobacco
500
0
0
0
0
0

aKanamycin resistant seedlings were histochemically stained for GUS activity. Wild type seedlings were not germinated on Kanamycin. One third of the kanR seedlings are double heterozygote for the 5'DGUS:Ac and 3'DGUS constructs or 5'DGUS:Ds and 3'DGUS. 3/4 are kanR in the selfed 3'DGUS T2 or selfed 5'DGUS:Ac seedlings.
bBlue sectors were detected in the root (R), hypocotyl (H), cotyledon (C) and first true leaves (1st).

Figure 1. Constructs used to monitor homologous recombination in various tobacco tissues. GUS transcription is driven by the 35S cauliflower mosaic virus promoter fused to the ? leader from tobacco mosaic virus. pGS001 and pGS008 or pGS009 were the recombination partners. pGS001 has a 500 bp deletion in the 3' end of the GUS gene, pGS008 and pGS009 have a 12 bp deletion in the 5' of the gene (*) which abolishes GUS activity. B= BamHI.


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