Download DNA methods for detecting and analyzing mutations in vivo

Mutation Research, 181 (1987) 227 234
227
Elsevier
MTR 04437
DNA methods for detecting and analyzing mutations in vivo
P.H.M. Lohman
a, j . V i j g b, A . G . U i t t e r l i n d e n
a n d F. B e r e n d s
b p. S l a g b o o m
b J . A . G o s s e n a.b
a
" TNO - - Medical Biological Laboratoo', P.O. Box 45. 2280 AA Rijswijk (The Netherhmds)
and h TNO
Institute for Experimental Gerontolo~,, P.O. Box 5815, 2280 H V R~jsuijk (The Netherlands)
(Received 25 February 1987)
(Accepted 11 May 1987)
Kevwor&." DNA methods; Mutations in vivo; Detection: Analysis; Aging, biological process.
The major adverse health effects in man attributable to exposure of somatic and germ cells to
carcinogens a n d / o r mutagens are tumor formation and, possibly, induction of heritable diseases.
In addition, the biological process of aging has
been suggested to be caused by the cumulative
effects of the continuous exposure to low levels of
in particular - - endogenous genotoxic agents.
The induction of damage in cellular D N A (adduct
formation, depurination, strand breakage) is considered as the initial step in the series of events
that leads to the above-mentioned adverse health
effects (Fig. 1). In this view, the subsequent step
would be the induction of mutations. Probably,
mutations predominantly result from so-called error-prone D N A repair, that is, the action of cellular repair systems that act on the induced D N A
damage and thereby alter the nucleotide sequence
of the D N A regions involved. The mutations resulting from this erroneous processing of induced
D N A lesions may comprise substitutions of one
nucleotide for another, deletions, insertions, D N A
rearrangements and various other types of changes.
When induced in certain crucial D N A sequences,
mutations may have severe biological consequences. For instance, they may prime the
processes of cancer induction (Reddy et al., 1982)
-
-
Correspondence: Prof. Dr. P.H.M. Lohman, Department of
Radiation Genetics and Chemical Mutagenesis, Sylvius
Laboratories, State University of Leiden, Wassenaarseweg 72,
2333 AL Leiden (The Netherlands).
and contribute to aging (Vijg et al., 1985a).
Sensitive techniques for the assessment of the
first step in the above described sequence of events
(the induction of D N A damage) have brought us
closer to (1) the firm association of adverse health
effects with suspected chemicals and (2) the estimation of the risk run by individuals possibly
exposed to genotoxic agents (Lohman et al., 1985).
Application of such recently developed techniques revealed that most chemicals induce a whole
spectrum of adducts in cellular DNA. These adducts can be repaired in different ways and with
different efficiencies. Moreover, both the induction and repair of a specific lesion can vary between different organs and tissues in the individual, and also as a function of the species or in
relation to age. Presently, sensitive immunochemical and biochemical methods are routinely used in
various laboratories, including our own, to establish the (age-related) organ- and tissue-specific
patterns of the induction and repair of various
forms of D N A damage in experimental animals
(Gupta et al., 1982; Poirier et al., 1983; Baan et
al., 1985; Lohman et al., 1985). In principle, a
similar approach can be followed to determine the
effect of exposures of human populations to
carcinogens.
In this way even the risk involved in exposures
to mutagens might be established were it not for
the lack of information about the relation between
D N A damage and the molecular endpoints at the
D N A sequence level in mammals. Information
002%5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
228
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Fig. l. Hypothetical pathw'ays via which induced D N A damage
may lead to adverse heMth effects.
about the induction and persistency of DNA lesions does not specify the harm that can be done
in terms of specific mutations. Therefore, an analysis of DNA damage and repair in vivo is especially valuable in combination with data on their
molecular consequences at the DNA sequence
level.
Mutation analysis in vivo
Thus far, studies on mutagenesis in mammalian
cells have been limited almost entirely to the in
vitro situation. It is not inconceivable, however,
that the spectrum of mutations induced in cells
that are removed from their natural environment
differs from that induced in the in vivo situation.
Indeed, both the induction of DNA damage and
their (attempted) repair are highly complicated
processes which are influenced by various regulatory systems, part of which are only present in
the intact animal. Especially established (immortal) cell lines can hardly be considered to
represent the in vivo situation as most cell lines
lose many specific properties upon culturing.
Moreover, since not all cell types can be cultured,
a comparative analysis of mutation induction in
cultured cells from various organs and tissues is
not possible. Therefore, the eventual system for
studying the relationship between DNA damage
induction, DNA repair and mutation induction is
necessarily the intact animal.
Recently, methods have become available for
studying somatic mutations in human and animal
cells in vivo. One of these methods is based on the
development of procedures for the enumeration
and clonal expansion of lymphocytes mutated at
the hprt locus in vivo (Albertini et al., 1982).
Apart from the specific cloning and culturing requirements, the principle of mutation detection
and characterization (by restriction-enzyme analysis of genomic DNA, isolated from expanded
mutant clones) is not different from that of in
vitro systems. The availability of this technology is
essential for comparing mutagenesis in vivo with
that in vitro. With respect to the occurrence of
spontaneous mutations in vivo, the first results
have appeared already. Data from Turner et al.
(1985) and Albertini et al. (1985) revealed that in
human T lymphocytes the frequency of 6-thioguanine-resistant cells is about 10 6-10 5 and
that a large part of the mutations involved are
major gene alterations (deletions, amplifications).
Turner et al. (1985) also pointed at the observed accumulation of mutations at the hprt locus
with age (Morley et al., 1982; Vijayalaxmi and
Evans, 1984; Trainor et al., 1984) which corresponds to earlier observations of an increased
frequency of chromosomal aberrations with age
(Brooks et al., 1973; Hedner et al., 1982; Evans,
t984). In view of the possibility that somatic mutations may be involved in the aetiology of aging,
it seems most relevant to analyse spontaneous
mutation induction in vivo in relation to age, in
order to obtain information about how such
changes can lead to the progressive deterioration
of cellular functioning. For extensively studying
mutation induction in vivo, the above-described
cell cloning technique is not sufficient, however.
Like its in vitro counterpart the method is too
time-consuming and it does not allow rapid and
detailed characterization of mutation. Furthermore, the method is limited to cell types that can
be cloned and cultured. It will be necessary to
search for mutation analysis assays that in principle are applicable to all human and animal tissues
subject to the phenotypic effects (cancer, aging
and heritable disorders).
Shuttle vector analysis in transgenic mice
Shuttle vectors are cloning vehicles that can be
propagated in both mammalian and bacterial cells,
229
Provided with a suitable target for mutagenesis
(for instance lac target sequences derived from the
M13 sequencing system), these systems are extremely powerful for the sensitive detection and
characterization of mutations (for a review, see
Lehman, 1985; Mekler et al., 1985). After the
rescue of the vectors from mammalian cells that
have been subjected to a mutagenic treatment, the
resulting mutated vectors can be selected and expanded in bacteria, which permits a subsequent
rapid characterization procedure based on restriction enzyme analysis and nucleotide sequencing.
A logical extension of the above-described studies on mutation induction at the hprt locus in vivo
would be to apply shuttle vector systems adapted
to the in vivo situation. This might be accomplished by the application of transgenic mice technology. In the ideal case, transgenic mice harbor
in all cells of their body a copy of an experimentally introduced DNA fragment (Palmiter and
Brinster, 1985). Such mice can be obtained by
injection of a few hundred copies of the particular
DNA sequence in one of the pronuclei of a fertilized egg and subsequent implantation of the egg
in the oviduct of a pseudopregnant mouse. The
efficiency of producing transgenic mice amounts
to about 25% when linear molecules are microinjected (Brinster et al., 1985).
A strain of mice harboring a shuttle vector
system for mutation analysis would be an ideal
tool for studying mutation induction in a specific
marker gene (bacterial or viral mutagenesis target)
in various organs and tissues of an experimental
animal (Fig. 2). Mutation induction can then be
studied following treatment with carcinogens
a n d / o r in relation to age. Such an approach would
introduce the advantages of shuttle vector systems
(rapid selection and characterization of the
mutants at the DNA sequence level in bacteria) in
the in vivo mutagenicity studies with mammals.
This would provide the possibility to comparatively study mutation induction in various organs
and tissues, in relation to the initial genotoxic
effects of DNA damage induction. It is even not
inconceivable that such an approach would circumvent the high background mutation frequency
of shuttle vectors that has often been observed for
mammalian cells in vitro (Razzaque et al., 1983;
Sarkar et al., 1984; Lebkowski et al., 1984). If the
fertilized egg
w i t h pronuclei
mjection
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v i r a l vector DhA c a r r y i n g JacZ
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• wild type
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Fig. 2. Mutation analysis in transgenic
bacteriophage lambda-derived shuttle vector.
mice.
using a
hypermutability of many shuttle vectors is not
inherent to their sequence but is a phenomenon
due to the vulnerability of the DNA during and
shortly after the introduction into the host cell, a
stable situation may be reached after integration
in the DNA of the egg. The only biological disadvantage of the transgenic mouse mutation model
would then involve the artificial target for mutagenesis. However, if the system can be developed
successfully, a similar approach to analyse mutagenesis of endogenous (naturally present) genes
may have come within reach.
Direct analysis of mutations in endogenous genes
Direct detection and analysis of mutant sequences in total genomic DNA is theoretically
possible by Southern blot restriction-enzyme analysis, because DNA probes are available for a
variety of human and animal sequences. Understandably, the crucial problems in such a direct
analysis are the low sensitivity (bands representing
mutants can not be detected in a Southern blot) in
addition to the limited number of mutations that
can be detected, namely those that are present in
restriction sites.
Recent developments in DNA diagnostics may
offer new possibilities. In 1979 Fischer and Lerman reported on a novel DNA separation technology which allows DNA fragments to be separated
on the basis of their base-pair composition (Fischer
230
and Lerman, 1979). Separation of DNA-restriction fragments by neutral gel electrophoresis is
based on the higher migration rates of small fragments in comparison to larger ones. Separation of
DNA fragments of the same size differing in their
base-pair composition is possible by using denaturing gradient gel electrophoresis (Fischer and
Lerman, 1980+ 1983). This is based on the fact that
partially melted DNA molecules have a different
migration in agarose
rID
"O
13
1
Fig. 3. Ethidium bromide-stained two-dimensional gel separation pattern of a mixture of the RsaI-digested plasmids
pOS184 and pOS185 (Mattern et al., 1985). The largest (2305
bp) Rsal fragment of pOS184 differs from the corresponding
fragment of pOS185 by a single base-pair substitution. A
mixture of the RsaI fragments of pOS184 and pOS185 was
fractionated according to size on 1% agarose (lst dimension).
The gel-lane was then attached to a 4% polyacrylamide slabgel
with a gradient of denaturant (0-32% formamide, 0-5.6 M
urea). After electrophoresis in the 2nd dimension, the single
agarose band was separated into two spots in the denaturing
gradient owing to a single base-sequence difference between
the two fragments. The sequence diversity suggested by the
similar behavior of the second largest Rsal fragments of pOS184
and pOSl85 is presently being investigated.
electrophoretic mobility in polyacrylamide gels
than their native equivalents (Lerman et al., 1984).
A transition of a perfectly double-stranded DNA
fragment to its partially melted form can be
achieved by polyacrylamide gel electrophoresis at
elevated temperatures in a denaturing gradient of
urea/formamide, parallel to the direction of
migration. It has been shown that these transition
points are highly sequence-specific. Control of
electrophoretic mobility by sequence specific melting equilibria provides a means for the separation
of DNA fragments differing only by single basepair substitutions (Fig. 3). Novel developments
have demonstrated that virtually all types of
single-base substitutions can be detected in a DNA
sequence of interest (by separation from the wildtype sequence), provided the sequence is attached
to a GC-rich sequence, a so-called GC-clamp
(Myers et al., 1985a,b).
Recently it has been shown that the denaturing
gradient technology makes it possible to detect
single base-pair substitutions in single copy genes
(Myers et al., 1985c) and to analyse sequence
diversity in repetitive sequences (Uitterlinden et
al., 1987). In another recent report, a general
method was presented in which denaturing gradient analysis was successfully applied for the separation, isolation and characterization of DNA
fragments, chemically mutated in vitro (Myers et
al., 1985d). Important advantages of mutation detection by denaturing gradient technology are evident: (1) It is possible to physically separate
mutant DNA fragments by preparative denaturing
gradient gel electrophoresis and to rapidly isolate
them in the absence of phenotypic selection and,
(2) in principle all types of mutations, including
single base-pair substitutions and small deletions,
can be detected in any cloned DNA sequence.
This will allow the direct detection of mutations in
the human population newly arising from generation to generation (Delehanty et al., 1986).
Mutation analysis using tongue clamp constructs
In collaboration with Dr. S.G. Fischer (Lifecodes Corporation, New York) we are presently
developing a system for direct mutation analysis
in endogenous genes (schematically depicted in
Fig. 4). The system is based on the use of so-called
231
4"
BaiH' EciR' "'°i'"
clamp
tongue
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- hybridization
- S 1 nuclease
- kinase/ligase
denatured genomic
digest
(Eco R I / H i n d III)
wild type sequence
mismatched (mutant)
,///////////////////~
II
seq. . . . . .
Polymerase Chain Reaction
amplification
~ preparative denaturing
gradient analysis
mutants
~c~
-,~-- wild type
cloning of mutants
Fig. 4. Detection and isolation of mutant DNA sequences by
using ' tongue-clamp' constructs.
tongue-clamp constructs, designed by Dr. Fischer
for diagnostic purposes. As shown in Fig. 4, a
tongue-clamp construct consists of a singlestranded D N A probe, specific for the fragment of
interest, attached to a double-stranded GC-clamp;
because of the GC-clamp virtually all mutations in
the fragment of interest will be detected (Myers et
al., 1985a,b). With an excess of a tongue-clamp,
specific for the fragment of interest, all copies of
this sequence can in principle be isolated from
(restriction-enzyme digested) denatured genomic
D N A by means of solution hybridization. Upon
renaturation, hybridization of most genomic D N A
fragments will be imperfect (as a consequence of
the presence of repeats). Such fragments are a
good substrate for Sl-nuclease, and can be removed in this way. (Since the same is true for
large mutations in the sequence of interest, this
method is only suitable for the detection and
analysis of small mutations.) The perfectly fitting
wild-type sequences as well as small mismatches
(due to base substitutions or small deletions) will
be saved after hybridization to the tongue-clamp.
Subsequent denaturing gradient electrophoresis of
the purified homologous fragments allows the separation of heteroduplex (mutated) DNA fragments from the homoduplex (unmutated) ones.
The sensitivity of this system is expected to be
about the same as that of a Southern blot, which is
about one single copy gene per genome. This
means that theoretically a mutation frequency of
about 10 - 6 should still be detectable. However, a
recently published method concerning restrictionenzyme analysis of the /~-globin gene allows for
the enzymatic amplification of specific sequences
up to 220000-fold (Saiki et al., 1985). When this
amplification step is included in the above-described mutation detection system, it is theoretically possible to detect one single mutant per
D N A sample, irrespective of the number of cells
the sample represents. Cloning of the mutated
DNA fragments will allow further analysis at the
D N A sequence level.
Mutations due to transposable elements
Denaturing gradient technology can also be
applied for the analysis of transposition type of
events. Such alterations, that occur at certain sites
of the genome with high frequencies, can in principle be detected with current restriction-enzyme
analysis. The problem, however, is to identify such
hypothetical sites in the DNA of the mammalian
genome. DNA sequences shown to be a source of
variability and which are able to exert influence
on gene expression are transposable elements
(Nevers and Saedler, 1977). This type of mobile
DNA sequences are well documented in prokaryotes, some lower eukaryotes, plants and
Drosophila, but it is by no means clear whether
such sequences tend to move from one site to
another in the genome of somatic cells of mammals
in vivo (Sankaranarayanan, 1986). This possibility,
however, should be considered and it has recently
been speculated that transposition events may be
involved in aging and age-related disorders such
as cancer (Sager, 1983; Vijg et al., 1985b).
A first selection of the sequences that may be
involved in D N A rearrangements in mammals is
provided by a novel cloning system allowing the
isolation of unstable sequences, which, due to
certain structural features, especially inverted rep-
232
etitions, could thus far not be cloned by using
regular (E. coli) vector-host systems (Wyman et
al., 1985). In this cloning system, hosts carrying
mutations in the recB, recC and sbcB genes are
used instead of rec + hosts. Such mutants have
reduced recombination efficiencies. Thus, mammalian DNA sequences that can be cloned in
recBC sbcB- strains and not in normal rec ~
strains may act as specific recombination substrates in the prokaryotic cell. Sequences selected
via this cloning system could be useful probes for
the detection of site-specific recombinations in
mammals. In this respect dispersed repetitive sequences, which are likely candidates for transposable elements (Nevers and Saedler, 1977), are
especially interesting. If such sequences appear to
be among the selected unstable sequences, they
can be used as probes in a two-dimensional analysis of the genome. That is, if total genomic DNA
is digested by a restriction enzyme to fragments of
a sufficiently small average size, which are then
subjected to electrophoresis in 2 dimensions (on
the basis of both length and base-pair composition) and subsequently blotted onto a filter, hybridization analysis can be carried out with the
above-described unstable DNA fragments as
probes. In this way specific unstable parts of the
DNA of various organs and tissues can be screened
for minute alterations with a much higher resolution than can be obtained with one-dimensional
sizing gels (Uitterlinden et al., 1987). Any consistent changes in the D N A sequence organization
in samples from organs/tissues revealed in this
way can be further characterized by isolating the
relevant D N A sequences from a 2-D gel, clone
them and subsequent analysis by conventional
methods.
Concluding remarks
Although many methods are currently successfully applied for detecting exposure to mutagens
and carcinogens, no method is yet available to
quantify an increased carcinogenic or mutagenic
risk in populations exposed to suspected agents.
Biological monitoring of human exposure will be
the first approach in this direction. However, the
ultimate aim is estimation of risk (eventually perhaps even personal risk), which current biological
monitoring methodology does not allow. Most
techniques for biological monitoring of genotoxic
chemicals are still under development; the whole
field is in its infancy. At this moment there is no
proof that any of the phenomena used as criterion
for exposure, not even, for instance, the presence
of specific DNA adducts, is relevant for an increase of the number of mutations in germ cells or
for the appearance of clinically verifiable tumors.
The methodology that we propose represents a
first attempt to study mutation induction in various organs and tissues of animals treated with
genotoxic agents. We anticipate that combining
molecular dosimetry of specific DNA lesions with
measurements of genetic effects in various organs
and tissues of the treated animals will give information on the relative importance of certain lesions for the induction of mutations.
Acknowledgements
We would like to thank Dr. S.G. Fischer, Lifecodes Corporation, New York, (U.S.A,), for collaborating in part of this study and for his helpful
discussions.
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