| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 89 === A filamentous phage Lf which infects Xanthomonas campestris pv. campestris, an important plant pathogen, has a circular single-stranded DNA genome of 6,008 nt. It is similar to the Ff phages that infect E. coli in terms of morphology and gene organization. Ten genes arranged as gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI-gXI have been found in Lf. The most abundant structural protein is the major coat protein encoded by gVIII. To isolate the promoter region of this gene, the 2 kb SacI fragment of Lf was partially digested with Sau3AI, or specific fragments within this region were amplified by PCR, and each individual fragment was subcloned into two promoterless vectors, pFY12-8MD1 and/or pFY13-9, respectively. The clones expressing lacZ in Xanthomonas campestris pv. campestris were sequenced. The results demonstrated that the promoter region encoded gVIII is mapped between Lf nt2,886-2,966. In an effort to examine the gene products of Lf, Northern analysis was performed using end-labeled oligonucleotides or double-stranded DNA probes specific to gV, gVII, gIX, and gVIII, respectively. Three species of mRNAs of size 870 nt, 320 nt, and 250 nt were detected with probes specific to gVIII, two species of mRNAs of size 870 nt and 320 nt were observed with either gVII or gIX and only one message of 870 nt was detected with a probe specific to gV. To identify the transcription initiation site, primer extension analysis was carried out. The results show that the transcripts start at the positions of Lf nt2,780, 2,807, 2,840, 2,880, and 2,922. The first three bands contribute to the 320 nt of mRNAs and the nt2,880, and nt2,922 bands denote the 250 nt of mRNAs shown on Northern analysis. Construct pG8PS containing nt2,886-2,966 still shows promoter activity, suggesting that the transcription initiation site of gVIII is at nt2,922, and the promoter for gVIII could be narrowed down at nt2,886-2,921. Within this region, however, no E. coli-type -35 and -10 consensus sequences of promoter were found. The most abundant transcript shown on primer extension analysis was the transcript initiated from nt2,880. However, no promoter activity could be identified near upstream of this region suggesting that this transcript could be a degradation product of larger mRNAs. The abundant transcript and the best fit of Shine-Dalgarno sequence for gVIII demonstrate that gene VIII has the advantage to generate a great amount of major coat proteins.
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