Whole seedlings were fixed in 1.5% (v/v) formaldehyde and 0.5% (v/v) glutaraldehyde made up in PEMT buffer(50 mm PIPES, 2 mm EGTA, 2 mm MgSO4, 0.05% [v/v] Triton X-100, pH 7.2) for 40 min and rinsed in PEMT buffer three times for 10 min. They were subsequently digested with 0.05% (w/v) Pectolyase Y-23 (Kikkoman) in PEM buffer (50 mm PIPES, 2 mm EGTA, 2 mm MgSO4) with 0.4m mannitol for 20 min, rinsed in PEM buffer three times,
treated with 220°C methanol for 10 min, and rehydrated in phosphate-buffered saline (PBS) for 10 min. Autofluorescence caused by free aldehydes from glutaraldehyde fixation
was reduced with 1 mg mL21 NaBH4 in PBS for 20min, followed by the treatment with 50 mm Gly in PBS(incubation buffer) for 30 min. Seedlings were incubated with primary antibodies against tubulin at room temperature overnight, rinsed in incubation buffer three times for 10 min, and secondary antibodies applied for 3 h at 37°C.After rinsing in PBS three times, root tips were cut off from the rest of seedlings, and mounted in 0.1% (w/v) paraphenylene diamine in 1:1 PBS-glycerol, pH 9. Cut cover glasses were used to space slide and cover glasses so as to avoid crushing delicate root tips.

Abstract:The structure and biological function of the DNA double helix are based on interactions recognizing sequence complementarity between two single strands of DNA. A single DNA strand can also recognize the double helix sequence by binding in its groove and forming a triplex. We now find that sequence recognition occurs between intact DNA duplexes without any single-stranded elements as well. We have imaged a mixture of two fluorescently tagged, double helical DNA molecules that have identical nucleotide composition and length (50% GC; 294 base pairs) but different sequences. In electrolytic solution at minor osmotic stress, these DNAs form discrete liquid-crystalline aggregates (spherulites). We have observed spontaneous segregation of the two kinds of DNA within each spherulite, which reveals that nucleotide sequence recognition occurs between double helices separated by water in the absence of proteins, consistent with our earlier theoretical hypothesis. We thus report experimental evidence and discuss possible mechanisms for the recognition of homologous DNAs from a distance.