| Objective: Bacteriophage is a kind of bacterial viruses, which created evolutionally intimate relationship with its host bacterium. The studies of bacteriophage genomics and functional genomics are obviously important to deep-looking insight into life origin, biotic diversity, organism evolution, genetic variation and metabolism and so on.The previous work of our group has figured out the whole genome sequence of Pseudomonas aeruginosa phage 3 (PaP3), which is dsDNA phage. PaP3 genome is composed of 44249bp, with 247 ORFs(>100bp) and 69 predicated genes, among which there are 6 genes respectively coding primase/helicase,two DNA polymerases, exonuclease, terminase large subunit, and lysozyme. The rest are functionally unknown. But these results are predicted by bioinformatics analysis software, and they must be further confirmed by experiments. Aim of present work is trying to prove experimentally which ORFs of the 69 ORFs are the true genes based on the mRNA or protein expression. Method: To prove which of the 69 ORFs is the true gene based on the mRNA or protein expression, following methods were performed: ①designed specific oligonucleotide probe based on the sequence of given ORF, and then labelled it with digoxin; ②infected the host bacteria with phage PaP3, then isolated and purified total RNA from infected bacteria at given time points; ③transcribe total RNA intocDNA with M-MuLV reverse transcriptase and used it as hybridized specimen; ④made dot-blots on nylon filter membrane with cDNA specimen, then hybridized with Digoxin-labelled ORF-specific probe to determine if there was expression of mRNA at given time points; ⑤isolated and purified phage PaP3 particles with PEG-sedimentation combined with CsCl gradient centrifugation, then the total proteins were isolated on SDS-PAGE to figure out how many proteins expressed; ⑥the protein bands on SDS-PAGE were transferred onto PVDF membrane by electronic transfer method. The 6 amino acids of the N-end of 90kD protein were sequenced by using Edman degradation method, and then clarified coding ORF of the protein from sequenced result.Result and Discussion: The results of dot-blot hybridization tests showed that the probe of ORF16912 gave positive result at 8h,and so did of ORF36894 at 1h to 8h.These results suggest both ORF16912 and ORF36894 are probably expressing genes. ORF16912 may express a later protein and ORF36894 may be a structural gene. However the probes designed from ORF9505,ORF18554,ORF40279,ORF41728,ORF42225 did not show positive. These results suggest three possibilities: first, these ORFs are not true gene; second, they are true genes but did not express in the conditions of present study; third, these ORFs expressed too little to be detected by the used method. The results of PaP3 particle protein on SDS-PAGE showed 9 bands, respectively with molecular weight 120kD, 110kD,90kD,55kD,50kD,35kD,34.5kD,33kD,24kD, suggesting that at least 9 genes of PaP3 genome expressed proteins.The protein bands in the electrophoresis gel were transferred onto PVDF membrane by electronic transfer method. The result of sequenced 6 amino acid residues at the N-end of 90kD protein showedthat there was not only unique at the first and second residues position, indicating the sequenced protein probably contaminated, but the residues of the third to sixth positions were unique. However the 6-residues DNRYLI, GNRYLI, ANRYLI, DKRYLI, GKRYLI, AKRYLI or 5-residues NRYLI, KRYLI and 4- residues RYLI reported at the N-end of 90kD protein were not found out from any protein coded by 69 ORFs or 247 ORFs. After the whole genome sequence of PaP3 was translated into protein, from which the 5-residues NRYLI was found in +1 frame, and 4-residues RYLI in -2 and -3 frames. However the molecular weight of the proteins with theses residues was smaller far from 90kD. The result suggests the sequence of reported protein be incorrect probably due to ①the protein amount on the PVDF membrane being too little to be sequenced; ②the specimen cont...
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