|本期目录/Table of Contents|

[1]姚蓉,A. J. L. Macario,E. Conway de Macario.马氏甲烷八叠球菌S-6的一个染色体区段的表达、序列分析和结构分析[J].应用与环境生物学报,1996,2(01):67-78.
 R. Yao,A. J. L. Macario,E. Conway de Macario.EXPRESSION,SEQUENCING AND STRUCTURE ANALYSIS OF A CHROMOSOMAL REGION OF METHANOSARCINA MAZEI S-6[J].Chinese Journal of Applied & Environmental Biology,1996,2(01):67-78.
点击复制

马氏甲烷八叠球菌S-6的一个染色体区段的表达、序列分析和结构分析()
分享到:

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
2卷
期数:
1996年01期
页码:
67-78
栏目:
论文
出版日期:
1996-02-25

文章信息/Info

Title:
EXPRESSION,SEQUENCING AND STRUCTURE ANALYSIS OF A CHROMOSOMAL REGION OF METHANOSARCINA MAZEI S-6
作者:
姚蓉1 A. J. L. Macario2 E. Conway de Macario2
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
Author(s):
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
关键词:
古细菌Methanosarcina mazei表达DNA序列分析DNA结构分析Southern杂交分析蔗糖梯度亚结构
Keywords:
archaebacteriaMethanosarcina mazeiDAN sequencingsequence analysisSouthern blot analysissucrose ultrastructure fractionation
摘要:
用一组多克隆抗体对马氏甲烷八叠球菌(Methanosarcina mazei)S-6菌株的基因组DNA文库进行了筛选。仅选出p60A克隆能对马氏甲烷八叠球菌中可发生细胞形态学变化菌株的抗血清发生阳性反应。对p60A克隆的双链DNA进行了序列分析。识别出一开式阅读框架(open reading frame P,ORF P).表达的蛋白是ORFP编码的蛋白的3倍,并得到ORFP蛋白的三聚体,在SDS-PAGE中表现出整体蛋白的迁移行为。同时,此表达蛋白抗10%SDS,6mol/L尿素及热处理。基因结构分析表明,ORFP具有与M.barkri mcr A有同源性的核糖体结合位点和一个与甲烷细菌启动子共有序列相似度达77%的启动子序列。使用参照序列分析表明,由ORFP演绎的氨基酸序列,具有高密度的带电荷的氨基酸和占优势的β-层迭构型。对此表达蛋白作了Neurosrora crassa的porin蛋白抗血清试验,以研究其可能功能。检验了M.mazei S-6细胞的蔗糖梯度制备物,对此原细胞中的ORFP蛋白作了定位。结果表明,ORFP蛋白可能是一种古细菌Porin,其在M.mazei S-6中的表达,可能象大肠杆菌那样与渗透压调节有关。
Abstract:
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.

参考文献/References:

1. Mah RA. Isolation and characterization of Methanococcus mazei. Carreat Microbiol. (1980)3:321~326
2. Mah RA,Kuhn DA. Transfer of the type species of the genus Methanococcus to the genus Methanococcus,naming it Methanococcus mmzei (Barker, 1936)comb. nov. et emend. and conservation of the genus Methanococcus(Approvel Lists, 1980)with Methanococcus varrielii(Approved Lists,1980)as the type species. Iat J Syst bacteriol. 1984,34:263~265
3. Robinson RW. Life cycles in the methanogenic archaebectera Methanococcus mazei. Appl Ervirom Microbiol. 1986,523:17~27
4. Yao R,Macario AJL,Conway de Macario E. Immunochemical differences among M, mazei S-6 morphologic forms. J Bncferid. 1992.174,4683~4688
5. Robinson RW,Aldrich HC,Hurst SF et al. Role of the cell surface of Methanococcus mazei in cell aggregation. Appl Raviroa Microbiol. 1985,49:321~327
6. Conway de Macario E. Macario AJL. Molecular structures of bacteria elucidated by monoclonal antibodies with special reference to antigenic determinants of the methanogens envelopes;In;Macario AJL.Conway de Macario E eds. Monoclonal Antibodies Against Bacteria. Vol Ⅱ.Orlando, Academic Press,1986.181~202
7. Conway de Macsrio E. Slide immunoenrymatic assay(SIA)in hybridoma technology.Methods is Eazymlolgy. 1986,121:509~525
8. Gander JE. Gel protein stains,Clycoproteins. Methods in Razymologg. 1984,104:447~448
9. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970,227:680~685
10. Segrest JP,Jackson RL. Molecular weight determination of glycoproteins by poiyacrylamide gel electrophoresis in sodium dodecyl sulfate. Methods in Eazymology. 1972,28:54~63
11. Towbin H,Cordon J, immunblotting and dot immunobinding-current status and outlook. J lmnuaol Methods. 1984,72:313~340
12. Towbin H,Staehelin T,Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellular sheets;Procedures and some applications. Proc Nall Acad Sci USA. 1979,76:4350~5354
13. Sanger F,Air GM,Barrell BG el nl.Nucleotide sequence of bacteriophage uX174. Nnture. 1977,265:687~695
14. Hsiao K. A fast and simple procedure for sequencing double stranded DNA with Sequenase. Nuc Acid Res. 1991,19:2787
15. Devereux P,Haeberli P,Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984,12:387~395
16. Maniatis T,Fritsch EF,Sambrook J. Molecular cloning,a laboratory manual. Cold Spring Harbor,Cold Spring Harbor Laboratory Press,1982
17. Boone DR,Whitman WB. Proposal of minimal standards for describing new taxa of methanogenic bacteria. Int J Syst Bacteriol. 1988,38:212~219
18. Paul C,Rasenbusch JP. Folding patterns of porin and bacteriorhalopsin. RMBT. J. 1985,4:1593~1597
19. Weil CF,Cram DS,Sherf BA et al. Structure and comparative analysis of the genes encoding component C of methyl coenzyme M raluctase in the extremely thenmophilic archaebacterium M.fernidus.J Bnrteriol.1988,170:4718~4726
20. Meile L,Stettler R,Banholzer R et al. Tryptophan gene cluster of M.thermoautotropkicum Marburg; nolecular cloning and nucleotide sequence of a putative try EGCFBAD operon. J Becteriol. 1991,173,5017~5023
21. Conway de Macario E,EVis EJ,Guzman R et al. Antibody-mediated activation of a defective B-D-galactosidase;Dimeric form of the activatable mutant enryme. Proc Nall Acad Sci USA. 1978,75:720~724
22. Stryer L. Protein conformation,dynamics and function. In:Biochemistry. New York:W. H. Freeman and Company,1988
23. Jap BK. Molecular design of Pho E porin and its functional consequences. J Mol Biol. 1989,205:407~419
24. Kleene R,Pfanner N,Pfaller R et al. Mitochondrial porin of N. crassa;cDNA cloning,in vitro expression and import into mitochondria.EMBO J.1987,6:2627~2633
25. Boone DR,Mat RA.Effects of calclum,magnesium,pH,and extent of growth on the morphology of Methanosarcira mazei S-6. Appl Envirom Microbiol.1987,48:127~136
26. Graeme-Cook KA. The regulation of porin expression in E. coli,effect of turgor stress. FEMS Microbiol Letters. 1991,79:219~224
27. Rampersand A,Utsuml R,Delgado J et al. Ca2+-enhanced phosphorylation of a chimeric protein kinase involval with bacterial signal transductlon. J Bacteriol Cbem. 1991.266:7633~7637
28. LiuY,Boone DR, Sleat R et al.M.mazei LYC,a new methanogenic Lsolate which praluces a disaggregating enzyme. Appl Ervirom Microbiol. 1985,49:608~613
29. Xun L,Mah RA,Boone DR. Isolation and characterisation of disaggregatase from M. mazei. LYC. Appl Eaniron Microbiol. 1990,56:3693~3698

相似文献/References:

[1]王振宇,余韬,张昱,等.采油废水缺氧处理体系细菌和古细菌丰度对比[J].应用与环境生物学报,2009,15(04):519.[doi:10.3724/SP.J.1145.2009.00519]
 WANG Zhenyu,YU Tao,ZHANG Yu,et al.Comparison of Eubacteria and Archaea Richness in Anoxic Treatment System for Oilfield-produced Wastewater[J].Chinese Journal of Applied & Environmental Biology,2009,15(01):519.[doi:10.3724/SP.J.1145.2009.00519]

备注/Memo

备注/Memo:
收稿日期:1995-5-5;接受日期:1995-9-25。
更新日期/Last Update: 1900-01-01