COMPARISON OF METHODS FOR EXTRACTING RIBONUCLEIC-ACID POLYMERASES FROM RAT-LIVER NUCLEI

被引:12
作者
BEEBEE, TJC
机构
关键词
D O I
10.1042/bj1830043
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 x 10(4) molecules of RNA polymerases I and III (altogether) and 1 x 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 x 10(4)-2.5 x 10(4) form-I and -III enzyme molecules (altogether) and a further 7 x 10(3)-8 x 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.
引用
收藏
页码:43 / 54
页数:12
相关论文
共 35 条
[1]   DNA-DEPENDENT RNA-POLYMERASE ACTIVITY OF CHINESE-HAMSTER KIDNEY-CELLS SENSITIVE TO HIGH CONCENTRATIONS OF ALPHA-AMANITIN [J].
AUSTOKER, JL ;
BEEBEE, TJC ;
CHESTERTON, CJ ;
BUTTERWORTH, PH .
CELL, 1974, 3 (03) :227-234
[2]   TEMPLATE SPECIFICITIES OF XENOPUS-LAEVIS RNA POLYMERASES - SELECTIVE TRANSCRIPTION OF RIBOSOMAL CISTRONS BY RNA-POLYMERASE A [J].
BEEBEE, TJC ;
BUTTERWORTH, PH .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1974, 45 (02) :395-406
[3]   TRANSCRIPTION FIDELITY AND STRUCTURAL INTEGRITY OF ISOLATED NUCLEOLI [J].
BEEBEE, TJC ;
BUTTERWORTH, PHW .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1977, 77 (02) :341-348
[5]  
BUHLER JM, 1976, J BIOL CHEM, V251, P1712
[6]   FACTOR STIMULATING TRANSCRIPTION BY RNA POLYMERASE [J].
BURGESS, RR ;
TRAVERS, AA ;
DUNN, JJ ;
BAUTZ, EKF .
NATURE, 1969, 221 (5175) :43-&
[8]   EUKARYOTIC NUCLEAR-RNA POLYMERASES [J].
CHAMBON, P .
ANNUAL REVIEW OF BIOCHEMISTRY, 1975, 44 :613-638
[9]   SELECTIVE EXTRACTION OF FORM I DNA DEPENDENT RNA POLYMERASE FROM RAT LIVER NUCLEI AND ITS SEPARATION INTO 2 SPECIES [J].
CHESTERTON, CJ ;
BUTTERWORTH, PH .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1971, 19 (02) :232-+
[10]   ANIMAL DNA-DEPENDENT RNA POLYMERASES .12. DETERMINATION OF CELLULAR NUMBER OF RNA-POLYMERASE B MOLECULES [J].
COCHETME.M ;
NURET, P ;
COURVALIN, JC ;
CHAMBON, P .
BIOCHIMICA ET BIOPHYSICA ACTA, 1974, 353 (02) :185-192