Recent Progress of Life Science Technology in Japan

305 Pages·1989·21.618 MB·English

RECENT PROGRESS OF
LIFE SCIENCE TECHNOLOGY
IN JAPAN
Edited by
Yoji Ikawa
Tsukuba Life Science Center
The Institute of Physical and Chemical Research
Akiyoshi Wada
Faculty of Science
The University of Tokyo
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Library of Congress Cataloging-in-Publication Data
Recent Progress of Life Science Technology
in Japan/Yoji Ikawa, Akiyoshi Wada [editors].
ISBN: 0-12-370652-1
Printed in Japan
89 90 91 92 9 8 7 6 5 4 3 2 1
Foreword
In June 1983 an intra-cabinet panel of the Japanese Government drafted a 10
year strategy for cancer control, recognizing the importance of this field of research.
The following month the Council for Science and Technology, the highest advisory
body to the Prime Minister in the field of Science and Technology, reported on "Fun
damental Strategies for the Promotion of Cancer Research."
On the basis of these two declarations, the Science and Technology Agency (STA)
of the Government in 1984 organized a research project titled "Research and De
velopment of a Fundamental Technology for Cancer Research" using the Special
Coordination Fund for Promoting Science and Technology. This was intended to
strengthen basic cancer research and to elucidate the underlying etiological factors
of cancer, as well as being broadly applicable to medically-oriented life science.
It is generally accepted that in modern biology the refining of each component
of a so-called bioscience triangle of DNA technology, protein technology and anti
body technology is requisite to furthering our understanding of the mechanisms of
cancer development and the essential nature of cancer cells, the ultimate goal being
to determine new methods which might successfully conquer cancer.
The above scientific research group was therefore organized to comprise two sec
tion - one concerning the development and evaluation of DNA technologies and
the other, protein-related technologies. Ten teams made up the former section and
14 teams the latter, with additional teams subcontracted making a total of 30. The
personnel involved were from industrial, academic and national laboratories and
brought diverse experience and viewpoints, thus enriching the overall potential of
the project. Accomplishments of all 30 teams during the first 3-year phase of the
program, ending March 1986, are summarized in this monograph and, in some cases,
findings through March of 1987 are included.
Highlights in the DNA-related technology section, headed by Professor Akiyoshi
Wada of the University of Tokyo, include the development of new automated equip
ment for DNA processing and sequencing, commercializing 'ready to use' acryla-
IX
X Foreword
mide gels for DNA sequencing and the development of an automated autoradiogram
reader. The protein-related technology section headed by the undersigned was suc
cessful in the fluorescein-labeling. of tryptophan in a peptide to microsequence ad
jacent amino acid residues, permitting easier estimation of DNA sequences for
synthetic DNA fragments to clone a gene encoding the peptide. This strategy was
applied to develop a super-micro amino acid sequencer capable of sequencing pep-
tides to the femto-mol order. In another area, new enzymes for manipulating pro-
teoglycans were isolated and characterized. These projects and other areas of research
are ongoing and details on them will be published in a subsequent report.
It is my firm belief that these newly developed and increasingly sophisticated tech
nologies and apparatuses will have many research applications in the broad fie Id
of life science. They will also be of great importance in our acquisition of insight
to the matter of carcinogenesis and, further, for the diagnosis, treatment and even
tual prevention of cancer.
I would like to take this opportunity to express my sincere gratitude to the ad
ministrative officers of the Life Science Division, Research Coordination Bureau, STA,
especially to Dr. Koichi Soga, Director of the Division, who have devoted great ef
fort to making the above research project a reality, and to the research scientists who
are actually participating.
June, 1989
Yoji Ikawa, M.D., & Ph.D.
Chairman,
Research Promotion Committee for
Research and Development
Preface
In the promotion of fundamental cancer research, the development and refining
of basic technologies for each component of the 'triangle of bio-sciences' -- DNA,
protein, and antibody - are essential, particularly in the elucidation of tumor-inducing
and tumor-suppressing genes, tumor-specific antigens, and so forth.
The first scientific research group of this project headed by Prof. Akiyoshi Wada
has attempted to introduce a systematic physical methodology of DNA analysis as
described by himself in the following pages, and has developed several automated
instrumentations for DNA sequencing. The achievement of this group are detailed
in part I of this monograph.
The second scientific research group of this project, headed by Dr. Yoji Ikawa,
has as its goal the refining of protein-related technologies for cancer research. This
group is working in three major subareas: (1) gene transfer and expression technolo
gies, (2) technologies for extraction, purification, and structural analysis of cancer-
related proteins, and (3) technologies for analysis and synthesis of saccharide chains.
Reports from these areas are respectively grouped in Part II, Part III, and Part IV
of this monograph. As for development of instrumentations for DNA sequencing,
we have received a number of useful suggestions from many biochemists and molecular
biologists. In organizing the second scientific research groups, we would like to thank
Drs. Ichiro Yahara, Michio Oishi, Tomoya Ogawa, Akira Kobata and Mitsuru Furusa-
wa for their thoughtful advices. Finally, we hope some of the technological infor
mation on cancer-related genes and proteins presented here will provide a meaningful
contribution to the advancement of basic cancer research.
Editors
xi
Preface
Concept of Machine-Aided Biophysical Research
The first half of this book contains reports of our efforts to introduce a systematic
physical methodology into DNA base sequencing study and innovative protein struc
ture analysis. The following is a summary of my concept of machine-aided biophysi
cal research and the background of how it has materialized. As everyone knows, a
living organism and thus its blueprint, DNA, contains a tremendous amount of in
formation which represents both its structural and functional features. Thus biolog
ical research ought to treat and process a large number of complex signals to obtain
a refined final picture. Modern scientific research begins with the extraction of data
from nature. These data should be as plentiful and as precise as possbile, so that
an accurate and detailed model can be constructed from them. In well organized
fields of physical science, such as particle physics, space science, material science,
etc. a variety of physical measurement methods have been adopted in strategically
well planned research projects, and these are playing a key role in the understand
ing of nature. By strategically well planned projects I mean those which are planned
very carefully from a broad perspective so that the object of the research, its method
and sequence, including sample or data processing, have been carefully examined
to assure the highest possible level of the entire project.
Biological research is, however, a bit behind research in other fields in terms of
strategic planning. One reason for this is the fact that biological organisms are very
comlex and unstable, and often provide non-reproducible results in a strict sense.
Thus it is hard to draw up a concrete plan of attack. But it is precisely this charac
teristic of biological organisms which necessitates the systematic planning of the en
tire series of studies in a "big" biological project such as human genome analysis.
Studying biological phenomena involves a series of operations: dissection, sam
ple preparation, physical and chemical measurements, information processing, model
building and database preparation. A characteristic of such a serial chain of opera
tions is that if one research unit in the chain is automated, resulting in increased
efficiency and accelerated processing speed, there is need to increase the processing
Xlll
XIV Preface
speed of the other research units. When the entire chain is viewed as a single sys
tem, this will result in the acceleration of research and the heightening of its quali
ty. One problem in biological studies has been the inability of many researchers to
gain an overall perspective of a project, since each research process demands sophisti
cated and sometimes tricky techniques.
I was troubled by this problem, and in 1981 began a study of the high speed
automation of DNA analysis using the Special Coordination Fund for Promoting
Science and Technology. One purpose of this study was to introduce the concept
of a well organized physical research style into biological processes such as DNA base-
sequence analysis, which requires extremely complex chemical and mechanical steps.
Another purpose was to prepare biological researchers and engineers in both the
government and private sectors for the 21st century, which promises to be an era
of massive reading of genetic information. This was the beginning of the automated
and high-speed DNA sequencing project in Japan.
The reactions to the proposed automation of DNA analysis by biologists in 1981
were quite interesting. A typical reaction was "why let machines do a job humans
can do?" My response was always "Why let humans do a job machines can do?"
Fortunately, I no longer hear such a reaction these days, and the concept of automa
tion and systematization in biological research has become acceptable to biologists.
Akiyoshi Wada
TEMPERATÜRE-GRADIENT DNA-PROBE COLUMN CHROMATOGRAPHY:
A NEW METHOD FOR DETECTION AND PURIFICATION
OF PARTICULAR DNAS OR RNAS
Akira Suyama
Hiromichi Tsurui
Mitsuru Yoneyama
Akiyoshi Wada '
Department of Physics
Faculty of Science
The University of Tokyo
Tokyo, Japan
2
Nobuyuki Baba
Scientific Instrument Division
Toyo Soda Manufacturing Co., Ltd.
Ayase, Japan
INTRODUCTION
DNA base sequences are somewhat like fingerprints and
unique to individual genes or genomes. As a consequce, the
sequences can be used to distinguish particular genes or
genomes from others. At the present time a fairly large
number of base sequences have been determined for various
genes and genomes which cause diseases, and the number is
Present address: The Technological University of
Nagaoka,Nagaoka 940-21, Japan
Supported by special research coordination funds from
the Science and Technology Agency.
Supported by grands from the Ministry of Education,
Science, and Culture.
RECENT PROGRESS OF LIFE SCIENCE 3 Copyright © 1989 by Academic Press/Harcourt Brace
TECHNOLOGY IN JAPAN Jovanovich Japan, Inc. All rights of reproduction in
any form reserved.
4 A. Suyama et al.
increasing. Therefore, a method which enables one to detect
particular base sequences would provide a good tool for (i)
the diagnosis of various diseases, including genetic disease,
cancer, and infectious disease, (ii) the detection of bacte­
ria in foods, and (iii) the screening or the purification of
particular DNAs and RNAs.
DNA-probes have been devised for the purpose of discrimi­
nating particular base sequences from others. Various
methods using DNA probes have been developed and have been
applied for the diagnosis of diseases, human chromosome
mapping, and screening of particular DNAs and RNAs(1-3).
Some of them are very powerful, and capable of detecting
single-base changes. Their detection procedures rely on
changes in restriction enzyme cleavage sites (4-7), on SI
nuclease (8, 9) or RNase A cleavage (10) of base mismatches
caused by single-base changes, on chemical modification of
base mis-matches (11), or on reduction in DNA duplex stabili­
ty caused by base mismatches (12-22). However, the procedure
always seems to be fairly complicated for non-experts, and at
least one day is required to finish the protocol. In addi­
tion, none of them could perform simultaneous discrimination
and purification, though this would supply very powerful
basic methods to molecular biology and genetic engineering.
For these reasons, we have developed a new method,
temperature-gradient DNA-probe column chromatography, which
provides accurate and reproducible DNA probing. The method
is easy and rapid (within two hours) to perform, and simul­
taneous purification of samples is available. Its sequence
resolution is extremely high so that single-base mismatch can
be detected. The present method is a kind of affinity chro­
matography using immobilized DNA probes so that simultaneous
discrimination and purification can be accomplished.
However, no former affinity chromatography using immobilized
nucleic acids (23-27) have succeeded in single-base mismatch
detection and rapid manipulation.
In the present report, we first describe the physical
basis of the equilibrium thermal stability of hybrid duplexes
formed between DNA probes and sample DNAs or RNAs. This is
required to establish the quantitative and high sequence-
resolution DNA probing method. Then we describe the princi­
ple, the instrumentation, the performance and the application
of our new DNA probe method.
Temperature-Gradient DNA-Probe Column Chromatography
II. THERMAL STABILITY
OF PROBE-SAMPLE HYBRID DUPLEX
A. Hybrid Stability
as the Basis of DNA Probing
DNA probes and sample DNAs or RNAs are capable of hybrid­
izing each other through the complementary base-pairing and
result in double-stranded hybrid formation. The strength of
DNA probe hybridization to sample molecules is determined by
the stability of the hybrid duplex formed. The other parts
of each sample molecule, which is usually longer than DNA
probe, remains on either side of the hybrid duplex and does
not affect the hybridization strength since they have no
interactions with the probe.
The formed hybrid duplexes may be melted into single-
stranded random coils by elevating temperature. Their
thermal stability under a given solvent condition is deter­
mined by three factors: (i) the G+C content and base sequence
of DNA probes, (ii) the length of hybrid duplexes, and (iii)
base mismatches in hybrid duplexes. The last factor allows
the detection of base sequence difference between DNA probes
and sample DNAs or RNAs using the thermal stability of the
hybrid duplex, and thus provides the basis of DNA probing.
B. Elimination
of Base-Pair Stability Difference
Tetraalkylammonium salt is useful for detecting the base
sequence difference between DNA probes and sample DNAs on the
basis of the thermal stability difference of the hybrid
duplex, since this salt eliminates the stability difference
between two complementary (A-T and G-C) base pairs (28, 29).
As a consequence, the thermal stability of the hybrid duplex
depends only on base mismatches created by the base sequence
difference, provided that DNA probes of the same length are
used.
In the case of DNA hybrid duplexes, the salt concentra­
tion required for elimination of the stability difference is
2.4 M for tetraetylammonium chloride (TEAC1) and 3.0 M for
tetramethylammonium chloride (TMAC1) (28). FIGURE 1 clearly
shows the elimination effect on the melting of long natural
DNA. The melting transition of ColEl DNA occurs over a wide
range of 15 °C in the absence of TEAC1 due to the stability
difference. In the presence of 2.4 M TEAC1, on the other