Metabolism and Enzymology of Nucleic Acids: Including Gene Manipulations

292 Pages·1989·13 MB·English

by  Hartmut Follmann

METABOLISM AND
ENZYMOLOGY OF
NUCLEIC ACIDS
Gene
Incl~ding
Manipulations
METABOLISM AND
ENZYMOLOGY OF
NUCLEIC ACIDS
Including Gene
Manipulations
Edited by
Jan Zelinka and Jozef Balan
Slovak Academy of Sciences
Bratislava, Czechoslovakia
PLENUM PRESS • NEW YORK AND LONDON
Library of Congress Cataloging in Publication Data
International Symposium on Metabolism and Enzymology of Nucleic Acids In
cluding Gene Manipulations (6th: 1987: Smolenice. Czechoslovakia)
Metabolism and enzymology of nucleic acids. including gene manipulations
edited by Jan Zelinka and Jozef Balan.
p. cm.
"Proceedings of the Sixth International Symposium on Metabolism and En
zymology of Nucleic Acids Including Gene Manipulations. held June 7-11.1987.
at Smolenice Castle. Czechoslovakia" - T.p. verso.
Sponsored by the Slovak Academy of Sciences.
Includes bibliographical references and indexes.
ISBN-13: 978-1-4612-8063-7 e-ISBN: 978-1-4613-0749-5
DOl: 10.1007/978-1-4613-0749-5
1. Nucleic acids-Congresses. 2. Nucleases-Congresses. 3. Methylation
Congresses. 4. Molecular cloning-Congresses. 5. Gene expression-Congress
es. I. Zelinka. J. (Jan) II. Balan. J. (Jozef) III. Siovenska akademia vied. IV. Title.
QP620.156 1987 88-39215
574.87'3282-dc19 CIP
Proceedings of the Sixth International Symposium on Metabolism and
Enzymology of Nucleic Acids. Including Gene Manipulations.
held June 7-11. 1987. at Smolenice Castle. Czechoslovakia.
This symposium was held under the auspices of the President of the
Slovak Academy of Sciences. Academician Vladimir Hajko.
© 1988 Plenum Press. New York
Softcover reprint of the hardcover 1s t edition 1988
A Division of Plenum Publishing Corporation
233 Spring Street. New York. N.Y. 10013
All rights reserved
No part of this book may be reproduced. stored in a retrieval system. or transmitted
in any form or by any means. electronic. mechanical. photocopying. microfilming.
recording. or otherwise. without written permission from the Publisher
OPENING OF THE SYMPOSIUM
I would like to open the Sixth International Symposium on Metabolism
and Enzymology of Nucleic Acids Including Gene Manipulations, organized by
the Institute of Molecular Biology of the Slovak Academy of Sciences under
the auspices of Academician Vladimir Hajko, President of the Slovak Academy
of Sciences.
This symposium, in a similar way as our previous meetings, is part of
the multilateral cooperation of the Academies of Sciences of the socialist
countries in the field of molecular biology and biochemistry.
We are glad to be able to welcome here, at Smo1enice Castle, numerous
outstanding scientists from many countries and our friends whom we tradi
tionally meet here.
Fourteen years have passed since our first symposium in 1973, and
tremendous advances have been achieved in the field of the metabolism and
enzymology of nucleic acids which have resulted in gene manipulations and
have stimulated the formation of modern biotechnologies. We are happy
that our Institute has contributed to this development in a modest way. Our
work was helped and supported by the organization of these symposia, giving
our younger co-workers the opportunity to find new stimulation for the~r
work and to get acquaint~d with scientists from other countries, thus fost
ering creative international cooperation. It is especially valuable that
this cooperation takes place between countries without regard to their
socio-economic setup. The scientists who meet here hold different political
opinions, but in spite of this, we are long-time friends, we respect and
support each other, and each of us contributes to the development of science
which serves all of mankind. This is our contribution to world peace and
mutual understanding.
Let me use this opportunity to thank all our foreign friends who have
contributed in the past or are at present contributing to our mutual sci
entific successes or are helping us to master new methodological approaches.
I would like to wish you a successful meeting and to express my sincere
hope that you will feel at home at Smo1enice Castle.
J. Zelinka
Corresponding Member of the Czechoslovak and
Slovak Academy of Sciences
Director, Institute of Molecular Biology,
Slovak Academy of Sciences
v
CONTENTS
DNA Methylation and DNA Methyltransferase in Wheat
H. Follmann. R. Schleicher and J. Balzer 1
The X-Ray Analysis of Ribonuclease Sa
J. Seveik. E.J. Dodson. G.G. Dodson and J. Zelinka 9
The Nature of Distribution of Ribonucleotide Reductases
H. Follmann. J. Harder and H.P.C. Hogenkamp 19
Cloning. Expression and Nucleotide Sequence of a Gene Encoding
a Second Thioredoxin from Corynebacterium nephridii
C.J. Lim. J.A. Fuchs, S.C. McFarlan and H.P.C. Hogenkamp 27
Thioredoxin from Streptomyces aureofaciens
M. Kollarova, E. Ka~ova, K. Szuttorova, H. Follmann
and J. Zelinka 37
Two-Dimensional IH-NMR ~nvestigation of Ribonuclease Tl and
the Complexes of RNase Tl with 2'- and 3'- Guanosine
Monophosphate
H. RUterjans, E. Hoffman, J. Schmidt and J. Simon 43
Nucleoside Phosphotransferase and Nuclease SI' two Enzymes with
Acylphosphate Intermediates, but Different Mechanisms
H. Witzel, A. Billich, W. Berg. O. Creutzenberg and A. Karreh 55
Probing the Mechanism of Enzyme Action through Specific Amino
Acid Substitutions
A.J. Wilkinson 63
Site-Specific Restriction Endonuclease Sau BMK I from Streptomyces
aureofaciens
J. Timko, J. Turna and J. Zelinka 65
The Effects of N4- and C5-Methylation of Cytosine on DNA
Physical Properties and Restriction Endonuclease Action
V. Butkus, S. Klimasauskas. L. Petrauskiene. Z. Maneliene,
L.E. Minchenkova. A.K. Schyolkina and A. Janulaitis 73
Regulation of RNA Synthesis in Yeast: The Stringent Control
of Ribosomal RNA Synthesis
L.W. Waltschewa and P.V. Venkov 79
In Vitro Study on Ribosome Degradation in Rat Liver
E.N. Nikolov and T.K. Nikolov 85
Specific Labelling in the Functional Domain of Elongation Factor
EF-Tu from Bacillus stearothermophilus and from Bacillus
subtilis
J. Jon§k. K. Karas and I. Rychlik 95
GTP Expense at Codon-Specific Translation and During Misreading
L.P. Gavrilova and D.G. Kakhniashvili 101
Site-Directed Mutagenesis of Elongation Factor Tu
A. Parmeggiani. E. Jacquet. M. Jensen. P.H. Anborgh.
R.H. Cool. J. Jon§k and G.W.M. Swart 109
A Site-Mutated rrsD Gene Expression Construct: Impairment of
Ribosomal Function
J. Sedla~ek. J. Smrt. J. Lakota. I. Rychlik. M. F§bry and
O. Melzochova 117
Expression of Prochymosin cDNA in E. coli
M. Fabry, P. Ka!par. S. Zadra!il. F. Kapralek and J. Sedla~ek 123
Control of the Cell Cycle Start by Protein Kinase Genes in
Saccharomyces cerevisiae
H. KUntzel. J. Lisziewicz. A. Godany. E. Hostinov§.
H.H. FHrster. M. Trauzold and H. Sternbach 127
Molecular Analysis of Mitochondrial DNA and Cloning of its rDNA
Fragments from Tomato Cell Suspension Culture
B. Hause. F. Baldauf. K. Stock. C. Wasternack and M. Metzlaff 131
Nucleotide Compartmentation During Synthesis of Cytoplasmic rRNA
and tRNA a~ well as Cytoplasmic rRNA and Mitochondrial rRNA
in Tomato Cell Suspension Culture
B. Hause and C. Wasternack 137
Intracellular Location of some Purine- and Pyrimidine
Metabolizing Enzymes in Plant Cells
C. Wasternack. R. Walther. K. Glund. A. Guranowski.
H. Tintemann and J. Miersch 143
Some Properties of Dinucleoside Oligophosphate Phosphorylase
from Euglena gracilis
A. Guranowski. E. Starzynska and C. Wasternack 149
Expression of Genes Coding for Novobiocin Biosynthesis and
Novobiocin Resistance in Streptomyces niveus
D.A. Ritchie. K.E. Cushing. P.G. Logan and J.I. Mitchell 155
Chromosomal Rearrangements Affecting the Regulation of Uridine
Phosphorylase Gene Expression in Escherichia coli
A.S. Mironov. S.T. Kulakauskas. V.V. Sukhodolets 161
Repeated Sequences in Chromosomal DNA of Streptomyces
aureofaciens 2201
A. Godany. P. Prista!. J. Muchova and J. Zelinka 167
viii
Stable Gene Duplication by "Puzzle" Shot Gun Cloning in
Inverted Repeats of a Streptococcal Plasmid
G.S. Steinborn 173
Different Types of Repetitive Elements in Mammalian Genome:
Cloning and Characterization of cDNA Copies of Transcripts
Containing Repetitive Sequences
A.P. Ryskov, P.L. Ivanov, O.N. Tokarskaya, A.V. Akulichev,
E.T. Jumanova, A.G. Jincharadze and L.V. Verbovaya 175
Nucleotide Sequence Analysis of the 5'-End of Chicken c-myb Gene
P. Urbanek, M. Dvofak, M. Travni~ek, P. Bartfin~k and V. Pa~es 183
Cloning and Characterization of Human Genes Expressed in Neural
Cells
S.A. Limborska, P.A. Slominsky, N.E. Maleeva and S.A. Korneev 191
Detection of a ~-Globin Intron Mutation in a ~-Thalassemic
Patient from Azerbaijan
S.A. Limborska, A.N. Fedorov, V.L. Bukhman and M.I. Prosniak 197
The Gene of the Yeast Ribosomal Protein L32
M.D. Dabeva and J.R. Warner 203
Structural Comparison of some Microbial Ribonucleases
C.P. Hill, G.G. Dodson, S.N. Borisova, A.G. Pavlovsky,
K.M. Polyakov, B.V. Strokopytov and J. Sevcik 213
Structure Bases for Nucleotide Recognition by Guanyl
specific Ribonucleases
A.G. Pavlovsky, S.N. Borisova, B.V. Strokopytov,
R.G. Sanishvili. A.A. Vagin and N.K. Chepurnova 217
Crystallization and Preliminary X-Ray Structural Studies of RNase
Thl from Trichoderma harzianum
K.M. Polyakov, B.V. Strokopytov, A.A. Vagin, S.I. Bezborodova
and S.V. Shlyapnikov 223
Three-Dimensional Structure of RNase Cz from Aspergillus clavatus
at 1.35 A Resolution
K.M. Polyakov, B.V. Strokopytov, A.A. Vagin, S.I. Bezborodova
and L. Orna 227
Amino Acid Composition and Influence of the Modification of some
Amino Acid Residues on the Catalytic Activity of the
Spleen Exonuclease
A.T. Bakalova and L.B. Dolapchiev 233
Binding of the Metal Ions in the Active Site of the Exonuclease
from Crotalus adamanteus Venom
R.A. Vassileva and L.B. Dolapchiev 237
Ribosomal Nuclease with Activity Towards Double-Stranded RNA
M.A. Siwecka, M. Rytel and J.W. Szarkowski' 245
Investigation of Immunoglobulin Light and Heavy Chain Genes
Responsible for the Synthesis of Antibodies in Hybridoma
PTF.02
S.M. Deyev, V.A. Ajalov, D.N. Urackov. A.G. Stepchenko,
F. Franek and O.L. Polyanovsky 251
ix
Regulation of Cytochrome P-450 Biosynthesis in Alkane Assimilating
Yeast
B. Wiedmann, M. Wiedmann, W.H. Schunck, S. Mauersberger.
E. Kargel and H.G. MUller 259
On the Mechanism of Reutilization of Nuclear RNA Degradation
Products: A Study in Vivo
D.D. Genchev 267
Immunological Characterization of DNA-Dependent RNA Polymerase(s)
of Spinach Chloroplasts
E. Brautigam and S. Lerbs 275
DNA Crosslinked by UV Light to the Nuclear Lamina is Enriched in
Pulse-Labeled Sequences
Z.I. Galcheva-Gargova 283
The Mechanism of Arabinofuranosyl-Cytosine Inhibition in
Undifferentiated Human B Lymphocytes
T. Spasokukotskaja, J. Taljanidisz, M. Benczur and M. Staub 289
Closing of the Symposium by J. Zelinka, Corresponding Member of
the Czechoslovak and Slovak Academy of Sciences, Director
of the Institute of Molecular Biology, Slovak Academy of
Sciences 297
Participants 299
Author Index 303
Subject Index 305
x
DNA METHYLATION AND DNA METHYLTRANSFERASE IN WHEAT
Hartmut Follmann. Roland Schleicher
and J8rg Balzer
Fachbereich Chemie. Abteilung Biochemie
der Philipps UniversitAt
D-3550 Marburg. Fed. Rep. Germany
Plant DNA is distinguished from the deoxyribonucleic acid of all other
eukaryotic organisms by its substantial amount of the modified base. 5-
methylcytosine (mCyt). mCyt is not detectable in most fungi and in invert
ebrates. it constitutes only up to 1% of the bases in vertebrate DNA. but
it contributes 5-8 mol% of the total base composition in average plant DNA.
Thus. one fifth to one third (20-35%) of the cytosine residues are modified
in all plants. and nearly all cytosines are replaced by the methylated
analog in some special plant DNA fractions.
This unique base composition of plant DNA was recognized more than
three decades ago [1]; however details of the distribution and formation of
mCyt in the plant kingdom have received little attention until recently and
specific functions are still unknown today. It is almost impossible to
conceive that all these'modified cytosine residues could serve as built-in
regulatory signals that determine gene inactivity or activity. the function
which has been demonstrated in recent years for the few mCyt residues
present in mammalian genes [2].
DNA methylation is a postreplicational event in which methyl groups
are transferred from S-adenosylmethionine onto specific cytosine residues
(mostly those in C-G sequences) of DNA; no cytosine modification occurs on
the mononucleotide level. The reaction is catalyzed by DNA methyltrans
ferases (EC 2.1.1.37) which have thus far been characterized almost exclu
sively from mammalian sources.
COOH COOH
~H-NH2 NH2 ~H-NH2
NNH)2 CH2 - N~CH3 CH2
CH2 O~N.!l CH2
oJ.-N + CH3-~-C~d.nin. I S-CH~d.nin.
I ONA
DNA
Cytosine SAM OH OH 5 -Methylcytosi ne SAH OH OH
We reasoned that the high amount of mCyt in plants requires an
efficient enzyme system which might differ from the DNA methylase found in
animal cells in activity and in specificity. and could thus provide insight
. 1
into the origin and functions of DNA methylation in plants. With the
exception of methylase activity in extracts from pea seedlings [3] and the
enzyme purified from the unicellular algae, Chlamydomonas reinhardii [4]
DNA methyltransferase has not been demonstrated in plants. Because the DNA
of the green algae does not exhibit plant-specific mCyt level (0.7%) it was
even uncertain whether the Chlamydomonas enzyme could be compared with that
of higher plants at all.
It is our aim to develop an in vitro system in which plant DNA methyl
ation can be simulated qualitatively and quantitatively. To this aim we
have purified to near homogeneity a highly active DNA methyltransferase
from wheat embryo and initiated an analysis of the distribution of mCyt
during wheat seed germination and in wheat DNA fractions of different
sequence complexity.
WHEAT DNA METHYLTRANSFERASE
Germinating seeds are a better source than green tissues for .the
characterization of the enzymes of nucleic acid metabolism in plants. DNA
methylase activity in germinating wheat embryo had been shown to increase
two- to threefold during and following the first round of DNA replication
(i.e., 15-20 hours after the uptake of water) but the enzyme was also
present in dry, ungerminated seeds [5]. Thus, unlike the enzyme of deoxy
ribonucleotide biosynthesis [6], DNA methylase is not confined to the S (or
G2) phase of a cell cycle. The preparation of intact cell nuclei from
wheat embryo by Percoll density gradient centrifugation provided a basis
for the purification of wheat DNA methyltransferase [7]. After separation
of tightly bound endogenous DNA on a column of DEAE cellulose the enzyme
was purified further by· chromatography on Sephadex G-75, Blue Sepharose,
and DNA cellulose. This enzyme preparation had a specific activity of 2
nmo1 CHs incorporation·h-L.mg-l and was enriched 300-fold over the cell
free tissue homogenate. It exhibited two protein bands on SDS poly
acrylamide electrophoresis gels, corresponding to M = 50,000 and 35,000.
Under native conditions, a molecular weight of 50,050 - 55,000 was estab
lished by centrifugation in sucrose gradients. DNA methylase from maize
seedlings could be obtained in a similar way.
The properties of wheat DNA methyltransferase are compared with those
of the enzyme from Chlamydomonas and of some mammalian proteins in Table 1.
It is obvious that the enzymes isolated from a unicellular and a higher
plant are similar, but differ strongly in molecular weight and in speci
ficity from the mammalian enzymes.
DNA methylation in plants vs. animals is not only characterized by a
much higher level of methy1cytosine but also by lower sequence specificity
Table 1. DNA Methy1transferases purified from mammalian and plant sources.
spec.activity
Source of enzyme "r specificity for DNA pmol.h-1.mg-1 ref.
calf thymus 130 000 ss>ds, hetero>homologous 600 15
rat liver 115000 5 000 16
mouse ascites cells 180 000 1 500 17
human placenta 135 000 2 000 J8
Chlamydomonas reinhardii 58000 ds >55, homo = heterologous 180 4
wheat embryo 55 000 ds >55, homo > heterologous 2 000 7
2