Expression of Filamentous Phage ψLf Gene I Encodingn a 48-kDa Protein Associated with Host Cell Membrane

• Main Authors: Chang, Kaung-Huey, 張光慧
• Other Authors: Tseng, Yi-Hsiung
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/55952036363135393567

碩士 === 國立中興大學 === 分子生物學研究所 === 86 === 　　Filamenous phage ΦLf specfically infects xanthomonas py campestris ,the causative agent of black rot in crucifers ΦLf contains a citcular single-stranded DNA genome of 6,008bp ,uses a replicative form RF as the intermediate of replication, and manifests a non-Lytic life cycle . Like Ff phages (fl,fd and M13), ΦLf has similar biological proerties and genomic organization i,e,in the order gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-. AII the open reading frames for ΦLf have already been identified in our laboratory ,except for orf440 which is located downstream from gVI . 　　The orf440 may be required for phage assembly ,because i) similar to the assembly protein of fl ,a membrane-spanning domain was found between aa 221 and 236 and ii) an ATP -birdngmotif is present in the N-terminal 97-aa region implying that the phage is assembled by ATP hydrolysis . This gene codes for pI (48kDa) detectable in the crude extract of the ΦLf-infected cells, Xcp20H(ΦLf), but not in the Xcp20h and mature phage particle by Western blot analysis using the antibody raised against the pI protein expressed in Escherichia coli. 　　To understand more about the cellular location of pI,the cell envelope of the ΦLf -iffected Xcp20H was fractionjaed by sarkosyl solubilization followed by western blot analysis .Results from these .Results from these experiments showed that the pI the Kf- infected host ,Xcp20h f ,is an inner membrane protein . Deletioe of the 3,-terminal part of I the intergenic region (IR) from the ΦLf genome reulting in murant phage H2785m,was found to cause loss of phage export . Therefore ,it is possobe that the ΦLf PI plays a role similar to that of the Ff pI required for phage assembly and export 　　The transcription of ΦLf gI was studiedd by Northern blkot analysis using a strand -speific DNA probe for gI mRNA ,but no signal mas detecred, In this study, a hairpin structure nt 4,627 and 4,657 in the downstream from g1 was found. Therefore, it is possible that φLf gI is co-transcribed with gIII and gVI till this putative transcription, similar to the case of Ff gI.