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Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

1996 01
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R. Yao A. J. L. Macario E. Conway de Macario
1. Wadsworth Cester for Laboratories, Research, New York State Department of Health;
2. Department of Biomedical Sciences, School of Public Health, the University at Albang, Albany, New York 12201-0509, USA
archaebacteriaMethanosarcina mazeiDAN sequencingsequence analysisSouthern blot analysissucrose ultrastructure fractionation

Methanosarcina mazei S-6 genomic DNA library was screened using a panel of polyclonal antibodies. The clone, termed p60A,reacted only to antiserum against Methanosarcina strains which undergo morphology conversion was chosen for this study. Both DNA strands or clone p60A were sequenced. An open readins frame P (ORF P)was identified. The size of expressed protein is three times as much as that of ORF P. which is confirmed by subeloning and expression of ORF P. The result showed that the expressed protein is a trimer of ORF P protein which has an assedated migration behavior on SDS-PAGE. Also,the expressed protein was resistant to 10% SDS, 6 mol/L urea and heat treatments. The gene structure analysis revealed that ORF P has a putative ribosome binding site homologous to that of M. barkeri mcr A,and a promoter sequence which showed 77 % sequence similarity to promoter consensus sequence of methanogen. The deduced amino acid sequence of ORF P was analyzed with reference sequences, and showed high density of charged amino acids and predominant β-configuration. The expressed protein was tested with antiserum against Neurospora crassa porin to demonstrate its possible function. Sucrose gradient preparations of M. mazei S-6 cells were tacted tolocalize ORF P protein in native cells. The results suggested that ORF P protein is a possible archaebecterial porin and its expression in M. mazei S-6 may be involved in osmotic regulation similar to that in E. coli.


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