A rapid clustering scheme for Vibrio specieswas developed based on the analysis of 16S rDNA, which compriseda group‐specific polymerase chain reaction (PCR) and restriction fragmentlength polymorphisms (RFLP) of the PCR‐amplified 16S rDNA. The group‐specificPCR, using a specific primer Vib1F, clustered the Vibrio speciesinto two large groups: Vib1F+ group and Vib1F– group.The PCR‐RFLP, using ScaI and BlnI, further dividedthese groups into smaller groups. Finally, 46 Vibrio speciesand eight related species could be clustered into 14 groups usingthis rapid clustering method. Seasonal dynamics of the Vibrio communityin Yoshimi Bay, Hibiki‐nada Sea, Japan, were examined based on therapid clustering method. In both seawater and sediments, the groupcomprised Vibrio parahaemolyticus, Vibrio alginolyticus, Vibriocampbellii, Vibrio carchariae, Vibrio harveyi and Vibrionatriegens and was predominant when the seawater temperaturewas above 20°C, whereas the group of Vibrio splendidus biotypeI and Vibrio lentus was abundant when the temperature wasaround or below 20°C.
Specificity of a DNA probe developed for some species of a Vibrio core group was examined using a fluorescence resonance energy transfer system. The probe was designed based on the sequence of a 16S rDNA fragment of Vibrio parahaemolyticus cloned in a plasmid, pTPRI. The 5'-and 3'-end of the probe was ligated with fluorogenic dyes. The specificity was examined against clonal 16S rDNA fragments and chromosomal DNAs extracted from different species of Vibrio. The 16S rDNA fragments, cloned from V. parahaemolyticus, Vibrio natriegens, Vibrio fischeri, Vibrio mediterranei, and Vibrio tubiashii, had from one to four nucleotide substitutions compared with pTPRI in the probe region. Probe hybridization with the clonal or chromosomal DNA fragments was monitored by determining the increase in fluorescence during PCR amplification. At the highest stringent condition, the probe hybridized only to identical sequences, but not to the sequences with substitution.
D RNA 3Yalpha (D3Yalpha), a defective (D) RNA 3 derived from the Y strain of cucumber mosaic virus (CMV-Y), was further characterized in combination with different helper viruses in the genus Cucumovirus. Interestingly, Nicotiana benthamiana plants inoculated with CMV-D8 and D3Yalpha developed systemic symptoms which were different from those induced by CMV-D8. To elucidate the potential effects of D RNA 3 on virus infection on the basis of the original combination of CMV-Y and D3Yalpha, a point mutation was made in the coat protein gene, which determined symptoms, of either CMV-Y RNA 3 (Y3) or D3Yalpha. Symptoms induced on N. benthamiana and N. tabacum plants, and semi-quantitative RT-PCR revealed that the ratio of RNA 3 to D RNA 3 was associated with the differences of symptoms in the leaf tissues. Furthermore, analysis of in situ hybridization suggested that there were spatial effects between coat proteins of Y3 and D3Yalpha in the infected leaves.
A defective (D) RNA 3 naturally generated from the Y strain of cucumber mosaic virus (CMV-Y) was characterized using a biologically active cDNA clone. Sequencing of the clone revealed that the D RNA 3, named D RNA 3Yalpha, was derived from CMV-Y RNA 3 and contained a 10 nt deletion in the 5' untranslated region and a 162 nt deletion within the 3a open reading frame. Co-inoculation of D RNA 3Yalpha with CMV-Y derived from in vitro transcripts facilitated propagation of CMV-Y containing D RNA 3Yalpha in Nicotiana benthamiana or Nicotiana tabacum plants. CMV-Y, however, did not produce deletion mutants upon serial mechanical passages in the plants.
A self-complementary decadeoxyribonucleotide, 5'd(GAAGFGCTTC)3', containing 5-fluorocytosine (F) in substitution for cytosine at the methylation site of DNA-cytosine methylase HhaI (MHhaI) has been synthesized. MHhaI was inhibited by the pre-incubation of the enzyme with d(GAAGFGCTTC).
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