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Developmental Neurobiology, edited by
Philippe Evrard and Alexandre Minkowski.
Nestle Nutrition Workshop Series, Vol. 12.
Nestec Ltd., Vevey/Raven Press, Ltd.,
New York © 1989.
Gene Mapping Techniques
Jean-Louis Guenet
Institut Pasteur, 75724 Paris Cedex 15, France
Very accurate gene mapping is essential in both man and laboratory mammals ( 1 3). Several techniques have been used over the last 50 years to localize mammalian
genes on the chromosomes of a given species. This chapter reviews these techniques, with special emphasis on the most recent ones that represent a true breakthrough in formal genetics.
CLASSICAL GENE MAPPING TECHNIQUES
AND THEIR LIMITATIONS
When two genes are linked they have a tendency to cosegregate during successive
generations. The closer the linkage, the more absolute is the cosegregation. This is
the fundamental principle of gene mapping, which has been successfully applied to
all species, including plants, over many years. In mammals such as humans and
mice, the continued discovery of marker genes scattered throughout the genome has
facilitated the mapping of new genes so that we now possess for these two species,
particularly the mouse, linkage maps that are far more detailed than those existing
for other mammals.
In the mouse, special matings can be set up, with appropriate stocks, to test for
possible autosomal linkage after two successive reproductive rounds: In general,
cross-back crosses are used, cross-intercrosses being reserved for studies in which
the viability or the fertility of the homozygous mutant under study is impaired. In
humans investigations concerning linkage are based on pedigree analysis.
In other words, for both species it is essential to define as a starting point a situation where two genes are heterozygous and either in repulsion A + / + B or in coupling AB/ + + , then to look for changes in this configuration after a reproductive
cycle (forms in coupling giving rise to forms in repulsion and vice versa), and finally to count the percentage or frequency of these recombination events. The
smaller the frequency, the tighter is the linkage.
At the population level other approaches have been used to test for possible linkage. One such method used in human populations is the lod score, which assesses
abnormally high frequencies of association between genes within populations hav87
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ing common ancestors. In the mouse the use of congenic strains, and especially recombinant inbred strains (RIS), has proved very useful. The use of RIS for the
detection of linkage has been extensively reviewed by Taylor (4), who has considered each strain as the equivalent of an F2 individual with a unique reassociation of
parental characters. Here, however, the new genotype is permanent since the strain
is inbred.
Although such classic techniques have been very useful over the years they share
several drawbacks and have in addition two intrinsic major limitations.
The drawbacks include their time-consuming nature, particularly in human studies; their expense, since several hundred mice may have to be bred to test for a hypothetical linkage or to ensure the necessary precision (since by definition as many
recombinants as possible must be scored if tight linkage is to be detected or established with accuracy); and the fact that many mutant genes cause reductions in viability or are not fully expressed in all individuals bearing them. This is particularly
likely to happen in the mouse and may prevent the determination of the true segregation ratio, and thus the proportion of recombinants in linkage crosses.
The two main limitations arise because: (a) The mapping methodology only allows for linkage detection with already known marker genes. By definition, linkage
cannot be detected if the gene under investigation is "far away" from all other
known genes, (b) The genes coding for invariant products, which by definition have
no allelic forms, cannot be mapped using these methods.
It is mainly for these reasons that nonsexual techniques have been developed by
mammalian geneticists over the last twenty years. These methods use somatic cells
of different species grown in vitro and artificially hybridized. Although fundamentally different, these new techniques are complementary to the classic ones and
should be considered as additions rather than alternatives to the classic mapping
technologies.
GENE MAPPING WITH SOMATIC CELL HYBRIDS
Our knowledge of the mammalian genetic map (and especially the human map)
has increased considerably during the past 15 to 20 years as a result of a number
of very important advances in cell genetics. Among these were the discovery of
human-rodent and rodent-rodent cell hybridization and the study of the segregation
of different chromosomes occurring in such hybrids; the development of chromosome staining techniques (the so-called banding techniques) which permit unambiguous identification of all human, hamster, and mouse chromosomes at the
metaphase stage; and the discovery of several hundreds of biochemical markers in
the different species which can be identified and characterized as electrophoretic
variants (the so-called electromorphs or allozymes, when the particular protein is an
enzyme).
At present, every human chromosome and every mouse chromosome has at least
one biochemical marker and often many more. These technical advances have per-
GENE MAPPING TECHNIQUES
89
mitted tremendous progress in mapping of both human (mostly) and mouse genes.
For rapid chromosomal assignment of human genes, a panel consisting of a collection of cloned human-rodent cell-hybrids containing specific combinations of human chromosomes can be used. The genes coding for proteins that are specific to the
human species can be identified by their product, using special biochemical assays.
In parallel, it is also possible to identify individual human chromosomes among the
chromosomal set of the cell hybrids. Thus genes coding for unique human markers
can be mapped to the individual human chromosome that matches its particular pattern in the hybrid panel. In some instances only one chromosome of a given species
(humans in general) is present in addition to the normal complement of the partner
species of the hybrid, so that a nonambiguous localization of the gene can be made
almost immediately.
In order to increase such gene assignments, the following efforts have been made:
(a) To characterize more gene products physically, chemically, and immunologically in order to distinguish clearly human from rodent products. Monoclonal antibodies, electrophoresis, and isoelectrofocalization techniques and study of thermal
stability of proteins have been of cardinal importance in this field, (b) To devise
methods for inducing gene activities which are not normally expressed in cell hybrids, (c) To construct additional hybrid panels containing new combinations of human, mouse, or Chinese hamster chromosomes, either as intact chromosomes or
partially rearranged with each other, (d) To develop sets of deletion hybrids each
containing only one chromosome of a given species, with more or less extensive terminal deletion.
More than 400 genes have been localized using these somatic cell hybrid techniques, and about 40 new genes are mapped each year. It is, however, impossible
with these techniques to localize genes coding for monomorphic gene products
(those which exhibit no variation between the partners of the cell hybrids); they are
also characterized by a certain lack of precision in gene localization due to the limited number of chromosomal breakages available. This results in most assignments
being less precise than those obtained using the classic techniques.
Finally it must be pointed out that genes whose products are not expressed in vitro
cannot be mapped using these techniques.
THE USE OF RECOMBINANT DNA PROBES IN GENE MAPPING
With the recent development of molecular DNA technology, new techniques
have become available for mapping genes. Again, these new techniques are complementary to those already discussed. They involve the use of DNA probes.
A DNA probe consists of a small DNA segment, containing a specific sequence
of the genome, which is used for hybridization at the molecular level with genomic
DNA. To be easily kept and prepared in large quantities for hybridization, the DNA
sequence of the probe is incorporated within an autoreplicative structure, either a
bacteriophage or a plasmid, prior to hybridization. To be easily recognizable (the
GENE MAPPING TECHNIQUES
90
primary role of a probe) the DNA sequence is labeled, usually with a radioactive
isotope.
Probes containing more or less extensive parts of specific genes are able to hybridize with the homologous segment of the chromosome carrying the particular
gene if used under appropriate technical conditions. Thus if a radioactive probe is
hybridized with a sample of DNA prepared from interspecific cell hybrids segregating for a given set of chromosomes it is possible to identify, at least roughly, where
the locus coding for this gene is located by matching the pattern of positive hybridizations with the pattern of chromosome presence or absence in the panel (Fig. 1).
These techniques represent an important advance in formal genetics because they
allow gene detection without requiring their expression. Mapping of the sequences
of several important structural genes has been performed using heavily labeled
cDNA probes and restricted (cut in small segments) genomic DNA from several
sources: human-mouse hybrid cell lines have shown the localization of the a globin
gene on human chromosome 16 and the assignment of the 3 globin gene complex to
human chromosome 11; human-chinese hamster hybrid cell lines have allowed the
assignment of the insulin gene to human chromosome 11 (5): mouse-chinese hamster hybrid cell lines have allowed the assignment of the cluster of genes coding for
3 interferon to mouse chromosome 4 (6).
In situ molecular hybridization constitutes another promising development in
gene mapping. When DNA probes are labeled with 3H with a high specific activity,
rather than with 32P, and hybridized to well spread mitotic metaphase preparations
of somatic chromosomes, attachment of the probe to homologous regions on the
chromosomes occurs (5). Again it is possible to determine where a given sequence
is located on the chromosomes of a given species after autoradiography and computation of the grain count.
These hybridization techniques have proven very useful in formal mammalian genetics. However, they have a few disadvantages: a gene cannot be mapped unless a
specific radioactive probe has been made available; in the genome of a species there
are frequently sequences partially matching the probe (e.g., pseudogenes, genes
coding for isoforms) which makes the recognition of a given sequence difficult; and
Probe
FIG. 1. Detection of syntenic relationships with cellular hybrids. The probe is localized on
chromosome n°10.
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since no use is made of sexual reproduction the process of meiotic recombination
does not operate and thus no precise assignment is possible for a given gene.
DNA PROBES AS GENETIC MARKERS
With the increasing number of studies being carried out on the structure of genomic DNA it has become quite clear that polymorphism at the level of DNA is much
more intense than it is at the level of transcribed and translated gene products. This
can easily be explained by what geneticists call silent mutations, such as changes at
the level of DNA resulting, for example, in the substitution of an ACA for an ACG
codon, both coding for the amino acid phenylalanine. Moreover, most DNA polymorphism goes undetected simply because the DNA is not translated into proteins.
According to a recent theoretical estimate no more than 1% to 3% of the genomic
DNA is actually translated. If on the other hand one assumes (which is almost certainly true) that the coding sequences are randomly spread over the entire genome it
is possible to use polymorphism at the DNA level as a source of markers.
This has been achieved by the use of so-called restriction endonucleases. These
particular enzymes have the property of cutting the DNA in a nonrandom manner.
They cut when a particular short sequence of 4 to 8 nucleotides is detected on the
DNA strand; each restriction endonuclease recognizes a specific sequence of nucleotides. It is thus possible with a given enzyme to cut an entire genome into segments
of various sizes (a few kilobase pairs in general); this dissection of the genomic
DNA into small pieces can be made on different samples with two or more enzymes. If two individuals differ by just one base pair in a restriction site (a mutation)
the size of the restriction fragment will be different. If these two individuals belong
to two different species, such as mouse and man, the restriction fragment length will
almost always be very different. This is the so-called restriction fragment length
polymorphism (or RFLP).
After DNA restriction, the DNA fragments can be separated according to their
size using gel electrophoresis. It is possible to recognize a particular segment of the
genome hybridizing to a radiolabeled probe either directly on the electrophoretic gel
or more frequently on a transfer replica of it. The tandem probe-restriction fragment
can be considered as a marker which is normally unique for the genome since the
probability of finding two sequences giving a restriction fragment of the same size
and having a similar affinity for a given probe is very small indeed.
With the so-called restriction polymorphism it is possible to recognize an individual homozygous for a RFLP from another who is heterozygous. After electrophoresis of restricted DNA from a homozygous individual all fragments hybridizing with
a radiolabeled probe will be of the same size, producing a single line. Two classes
of labeled fragments will be recognized on DNA from a heterozygous animal, giving two lines.
Another advantage of RFLP is that the sequence which is used for the recognition
of a particular fragment does not necessarily code for a protein. It is not even obligatory for the sequence to be expressed; any "anonymous" segment of DNA, pro-
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vided that it is present in the genome under the form of a single copy, will do. About
1% of the recombinant phages in the human genomic library contain only unique sequences. Chromosomal localization of such unique sequences is then possible using
the techniques already reported (e.g., hybrid cells, in situ chromosome hybridization). It is also possible using conventional markers in conjunction with normal sexual reproduction. Together with Robert and co-workers (7) we have mapped the
gene coding for the light chain of myosin very close to the mouse Idh-1 locus (chromosome 1) using sexual reproduction (and thus meiotic recombination) and two different mouse species: Mus musculus domesticus (the normal laboratory mouse) and
Mus spretus (a line derived from a wild population of mice). These two lines, which
have been separated for millions of years, have lost mutual sex appeal. They no
longer interbreed and behave like two different species. In the laboratory, however,
interbreeding is possible. This allows a tremendous amount of polymorphism to
segregate since the genetic divergence has produced many translated or untranslated
changes at the DNA level. What we did originally, using a known a actin probe,
can now be generalized to any fragment, even an "anonymous" one. Once the
DNA fragment contained in the probe has been mapped to a given chromosome the
information is permanent and can, for instance, be used as a marker for other fragments. Data are cumulative and fresh information is now accumulating at an exponential rate. Avner and co-workers have used a similar approach for the study of a
particular part of the genome. After preparing a DNA library from an almost pure
preparation of mouse X chromosomes sorted by flow cytometry (8), X-chromosome-specific DNA probes have been localized using these probes at the molecular
level in conjunction with studies on animal phenotype using classical marker genes
for X chromosomes such as Tabby (Ta), jimpy (jp), a galactosidase (a gal), and hypoxanthine phosphoribosyl transferase (HPRT).
All these probes have been linearly arranged and can now be used to map further
sequences. It is obvious that the system is autocatalytic. It can also be generalized
to any chromosomes of a given species so that having a marker every milli-Morgan
is now a reasonable perspective. These molecular approaches to the mapping of
mammalian genes are universally applicable. For example, no understanding of the
biochemical nature of a given inherited disease is required for mapping its genetic
determinant. They open new and very promising perspectives in terms of genetic
counseling, as most common genetic diseases will probably soon become detectable
in carriers and pregnancies at risk identifiable. In the mouse, conventional mapping
techniques, although still in use even in the more advanced laboratories, are going
to be irreversibly transformed.
REFERENCES
1. Cooper DN, Schmidtke J. DNA restriction fragment length polymorphisms and heterozygosity in the
human genome. Hum Genet 1984;66:1-16.
2. Green MC. Gene mapping. In: The mouse in biomedical research. New York: Academic Press,
1981:105-17.
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3. Puck TT, Kao F-T. Somatic cell genetics and its application to medicine. Annu Rev Genet 1982;
16:225-71.
4. Taylor BA. Recombinant inbred strains: use in gene mapping. In: MORSE MC, III, ed. Origins of
inbred mice. New York: Academic Press, 1978:423-38.
5. Malcolm S, Barton P, Murphy C, Ferguson-Smith MA. Chromosomal localization of a single copy
gene by in situ hybridization—human beta globin genes on the short arm of chromosome 11. Ann
Hum Genet 1981 ;45:135-41.
6. Naylor SL, Gray PW, Lalley PA. Mouse immune interferon (IFN-gamma) gene is on chromosome
10. Somatic Cell Genet 1984;10:531-4.
7. Robert B, Barton P, Minty Y, et al. Investigation of genetic linkage between myosin and actin genes
using an interspecific mouse backcross. Nature 1985;314:181—3.
8. Baron B, Metezeau P, Kelly F, et al. Flow cytometry isolation and improved visualization of sorted
mouse chromosomes. Exp Cell Res 1984;152:220-30.
9. Taylor X, Bailey X. Proc Nad Acad Sci (USA).
DISCUSSION
Dr. Devilliers-Thiery: Do you have any idea how polymorphic genes are? In other words
what is the percentage of homology in terms of nucleotide sequences between two strains of
mice?
Dr. Guenet: Between two human beings one base per thousand per chain is different, so
you probably have 1,000 bases that are different from your neighbors'.
Dr. Devilliers-Thiery: To be able to map a variant gene, you have to have polymorphism.
Have you ever encountered a situation in which you had no polymorphism?
Dr. Guenet: No. There are so many restriction enzymes that the probability of not finding
polymorphism within an interspecific cross is very close to zero. You must bear in mind that
as well as the huge number of restriction enzymes you can use methylation, either independently or in addition, to change the restriction site. This gives you a tremendous advantage
compared with formal genetics.
Dr. Devilliers-Thiery: How close is the genetic map of the mouse to that of the human? In
other words, when you have mapped the mouse what are the chances that the human map will
be the same in terms of linkage for invariant proteins? If you know that in the mouse genome
gene A is next to gene B which is next to gene C, what are the chances that the same order
will also be found in the human genome?
Dr. Guenet: Invariant proteins have a tremendous degree of homology. Thus a probe obtained from the mouse will have a very good chance of matching the homologous human
gene. It is another matter for variant proteins, of course. As far as order is concerned, Taylor
and Bailey (9) recently reported that there are sequential homologies between the mouse map
and the human map. For example, chromosome 4 on the mouse map is almost a replicate of
the branch q of human chromosome 1. So if you find your receptor to be in chromosome 4 in
the mouse I would recommend that you look in branch q of human chromosome 1. There are
exceptions of course, but there are a large number of homologies.
Dr. Campagnoni: Can you map genes within a chromosome by taking somatic cell hybrids
that contain, for example, one human chromosome in a background of mouse chromosomes?
Dr. Guenet: Yes, this has been done. Attempts were made to map myosin with this technique, but the results were incorrect because several rearrangements caused confusion. When
multiple rearrangements occur the segments of the human chromosome may become so small
that they are no longer easy to identify.
Dr. Sotelo: You talk about hybridization in situ. If I understood you correctly, you have a
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cDNA probe and you use a single chromosome from one cell and try to find where the cDNAs
hybridize. Is it necessary that the gene is expressed?
Dr. Guenet: No. The big advantage of in situ hybridization and of all nuclear techniques is
that the gene does not have to be expressed. For example, the gene for phenylalanine hydroxylase is not expressed in vitro but, nevertheless, you can detect where this gene is located. This is not the problem with these techniques. The problem is the large amount of
background "noise". You have to count the grains and make the map by a process of addition.