Ghanim et al 1998 - Virology .pdf

302 GHANIM ET AL.V61 (nt 61–80, viral strand, 5ATACTTGGACACCTAAT-GGC3) and C473 (nt 473–457, complementary strand,5AGTCACGGGCCCTTACA3). Oligonucleotides werepurchased from Biotechnology General (Rehovot, Israel).The cycling protocol (using a Techne PHC-2 thermocycler)was as follows: initial denaturation for 3 min at95°C, annealing of primers for 1 min at 45°C, addition of1 unit of TaqI polymerase, extension for 2 min at 72°C,and denaturation for 1 min at 94°C; subsequent cycleswere: 1 min at 45°C, 2 min at 72°C, and 1 min at 94°C;after 30 cycles, the reaction was terminated by a 10 minincubation at 72°C (Navot et al., 1992). The PCR productswere subjected to electrophoresis in a 1% agarose geland were photographed. The amplified virus DNA wasidentified after blotting and hybridization with radiolabeledplasmid pTYH19 containing a full-length copy ofthe TYLCV genome (Navot et al., 1991). Autoradiographywasfor1to5h.Detection of TYLCV DNA in plants and in insects bysquash blot and Southern blot hybridizationSquashes of tomato leaves were prepared and hybridizedwith a radiolabeled DNA probe as described previously(Navot et al., 1989). Total DNA extracted from tomatoplants and from insects (egg, instar, and adult) was hybridizedwith radiolabeled plasmid pTYH19 as described (Czosneket al., 1988b; Zeidan and Czosnek, 1991).Dissection of ovaries and maturing eggs of B. tabaciand observation in the scanning electron microscopeInsects were kept on the microscope cylindrical mountusing double-sided adhesive tape. The abdomen wasseparated from the thorax and pressing the tip of theabdomen expelled its content. The internal organs werewashed several times with water using a Pasteur pipettewith a narrow tip, and the ovaries were separated fromthe other organs. For PCR analysis, DNA was extractedas described above. For microscope observation, thetissues were processed essentially as described by Nation(1983), omitting the treatment with hexamethyldisilazane.Ovaries were fixed by immersing the tissues in0.2% glutaraldehyde, 4% paraformaldehyde in PBS for 5min. The tissues were then flushed two to three timeswith sterile distilled water and allowed to air dry. Thesamples were observed in a JOEL 5410 LV scanningelectron microscope at low vacuum.Oviposition on artificial mediumViruliferous insects were placed in a 5-ml glass vial(one insect per vial). The vial was covered with a layer ofKimwipes held in place with a layer of stretched Parafilmmembrane (the paper allowed the eggs to stick). About0.5 ml LB medium containing 15% sucrose was depositedon the membrane and covered with a second layerof stretched Parafilm membrane.ACKNOWLEDGMENTSThis work was supported by Grant 95-168 from The US-Israel BinationalScience Foundation. M.Z. was recipient of The Golda Meir FellowshipFund.REFERENCESAl-Musa, A. 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