SPECIAL ARTICLE
Mammalian Somatic Hybrids and Human Gene Mapping
RAJU S. KUCHERLAPATI, Ph.D., and FRANK H. RUDDLE, Ph.D., New Haven, Connecticut.
A New York University Honors Program Lecture
Somatic cell hybridization, as an aid in the study of human
map, has made large strides during the past several years.
Rodent-human hybrids are readily obtained, and selective
systems are available to select hybrid cells to the exclusion
of parental cells. Various species-specific genetic markers
can be studied in the hybrids, and correlation of the
expression of these markers with each other and with specific
human chromosomes retained allows synteny analysis and
chromosome assignment. Methods to determine the relative
order of genes and their regional localization are also
available. Using these methods, more than 60 human genes
have been assigned to 22 human chromosomes.
H YBRIDIZATION of mammalian somatic cells and the analysis of the resulting hybrid cells have yielded valuable
information about various biological problems and promise future revelations into the nature and regulation of
gene action in mammals. One such area is gene mapping
in man. The earlier methods of gene mapping, primarily
sexual in nature, have provided important linkage information in the house mouse, Mus musculus, but proved to be
of limited value in human gene mapping. The advent of a
parasexual method of gene mapping provided a great
impetus to the development of human cells as a favorable
genetic system for investigation. The use of the parasexual
system for human gene mapping came mainly through four
advances: namely, [1] the ability to fuse cells from different mammalian species, [2] development of selective systems based on intergenic complementation, modeled after
microbial systems, which enables the preferential isolation
of hybrid cells to the exclusion of parental cells, [3] the
availability of various new biochemical and other markers
that can be assayed in cultured cells, and [4] the invention
of staining methods that result in linear differentiation of
mammalian metaphase chromosomes. We will briefly
present the rationale, methodology, use, and significance
of such mapping procedures in this report.
Hybrid cells derived from fusion of cells from various
animals have been described (for a catalog, see Reference
1), but we shall restrict our attention to one class of hybrid
• From the Department of Biology, Yale University, New Haven, Connecticut.
cells, those between rodent and human. The first report of
in-vitro hybridization of somatic cells was that of Barski,
Sorieul, and Cornfert (2), who showed that mouse cells of
high and low tumerogenicity were capable of fusion and
of forming viable proliferating hybrid cells. Ephrussi and
associates (3) realized the potential of this observation in
the development of a parasexual system for a genetic
study of mammals. This observation has been confirmed
and extended to crosses between cells from different
species (4, 5). It is of interest to note that incompatibility,
which is the hallmark of sexual fusions between species,
does not exist in these crosses. The ability to isolate stable
drug resistant cell lines (6) paved the way for the development of the first selective system to isolate hybrid cell
lines (7, 8). Okada (9) has discovered that Sendai virus, a
member of the parainfluenza group, is capable of mediating fusion. Use of this virus after the development of
effective inactivating methods, which inhibit viral replicaton without impairing the fusing ability of the virus (10),
make it possible to obtain hybrids with relative ease.
Weiss and Ephrussi (11) observed that the hybrid cells
derived from fusion of cells from different species segregate chromosomes, albeit slowly, and that such segregation
is unilateral. The major breakthrough in somatic cell
genetics was the discovery by Weiss and Green (12) that
mouse-human hybrid cells rapidly, preferentially, and
extensively lose human chromosomes. At the time, it was
known that the genomes of the two species were functional
within a common environment, as shown by the study of
lactate dehydrogenase expression in rat-mouse hybrids
(13). Weiss and Green showed that this phenomenon
holds true in mouse-human hybrids as well. If the human
chromosomes can be unequivocally identified, the ability
to distinguish between rodent and human gene products
and the correlation of the presence of a specific chromosome with a specific gene product enables assignment of a
gene to a chromosome. Although no good methods to
identify individual human chromosomes were available,
Weiss and Green found a consistent correlation for the
presence of a small human chromosome with the expression of the gene for thymidine kinase. The advent of
chromosome banding methods, primarily due to Caspersson and colleagues (14), provided a reliable means of
identifying individual mammalian chromosomes. As a
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553
result of these diverse developments, the area of human
gene mapping by somatic cell hybridization has blossomed.
Formation of Hybrids
Although hybrid cells can be recovered by mixing the
two desired cell types and cocultivating them, a fusion
promoting agent is usually added to the mixture. Various
agents have proved effective in enhancing hybrid cell
formation, including Newcastle disease virus (15), lysolecithin (16), pederine (17), and polyethylene glycol (18).
The most commonly used, if not the most effective agent,
however, is the hemagglutinating virus of Japan, otherwise
known as Sendai virus. Okada (9) and later Harris and
Watkins (19) showed that Sendai virus is capable of
agglutinating mammalian cells at 4 °C, and, when transferred to 37 °C, some of the cells undergo fusion. The
virus seems to cause cytoplasmic bridges between cells,
which result in fusion and heterokaryon formation. Experiments by Yerganian and Nell (20) and Coon and
Weiss (21) proved the usefulness of this system in producing viable, proliferating interspecific hybrids. Investigations
into the nature of the fusion agent showed that it is a
glycoprotein (for a review, see Reference 15).
Conditions of Hybridization
The effect of various determinants on the fusion and
recovery of hybrid cells has been studied extensively, and
these studies provided information regarding the optimal
conditions for hybrid cell recovery.
Croce, Koprowski, and Eagle (22) studied the effect
of pH and different buffering agents on hybrid cell
recovery. They have shown that a pH of 7.8 to 8.2 before,
during, and for a short period after hybridization results
in maximal hybrid yield.
The stage of the cell cycle where the two cell types are
at the time of fusion seems to have a profound effect on
hybrid recovery. It has been shown that cells in Gl, if
fused with cells at M stage in mitosis, result in premature
condensation of the Gl cell chromosomes, sometimes
causing pulverization of chromosomes (23). This phenomenon, found to be useful in the study of chromosome
structure and mitosis, is of limited value for study of
proliferating hybrids because none can be obtained.
The nature of the cell types used in hybridization also
has an effect on hybrid cell formation and recovery. For
example, the original hybridization experiment by Harris
and Watkins (19) between mouse Ehrlich ascites tumor
cells and human HeLa cells yielded heterokaryons but
failed to produce mononuclear hybrids. Johnson and
Harris (24) later showed that this was due to the suppression of DNA synthesis in the HeLa cell nuclei. Even
though such observations are limited, no general conclusions can be drawn about the nature of the cell types,
which when fused yield viable hybrids.
The hybrid recovery is not dependent upon the permanant versus nonpermanant nature of the cell type, but
crosses between two nonpermanant cell lines result in
hybrids whose survival rates are comparable with the
parents, whereas crosses in which at least one of the
parents is permanant result in permanant hybrids.
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Klebe, Chen, and Ruddle (25) studied the effects of
various input multiplicities on hybrid yield. Using two
different mouse cell types, they have shown that under
conditions in which both the parental lines can be selected
against, a 1:1 multiplicity gives optimal results. In cases
where only one of the parents can be selected against, a
higher ratio of selectable to nonselectable parent is recommended. It has to be realized, however, that under these
circumstances there might be a greater propensity of two
selectable cells fusing with one nonselectable cell, resulting
in cells with higher chromosome numbers and perhaps
with different properties.
Selective Systems
Because the fusion process does not involve all the cells
in the mixture and not all hybrids are the result of fusion
of unlike cells, it becomes necessary to enrich for hybrid
cells. There are a number of selective systems available
to achieve this goal, and most of them are based on conditional lethality. A few of them are described below.
The earliest system used for selection is visual. This
stemmed from the observation that some hybrid cells
seem to grow more vigorously than either of the parental
cells. If the fusion mixture is plated at low density, the
hybrid colonies grow faster, thus becoming visible earlier
and facilitating isolation. Differential growth, however, is
not the rule in all hybrid combinations, thus making this
method unreliable.
Drug resistant mutants are most widely used in selection systems. One such is the so-called HAT selection
system (hypoxanthine, aminopterin, and thymidine).
Folate metabolism plays an important role in purine and
pyrimidine nucleotide biosynthesis. This pathway can be
inhibited by the drug aminopterin. Wild type cells can
survive in medium containing aminopterin if exogenous
nucleotides in the form of hypoxanthine and thymidine are
supplied to the cells. Hypoxanthine is converted to inosine
monophosphate by a reaction mediated by the enzyme
hypoxanthine phosphoribosyl transferase and thymidine to
thymidine monophosphate by thymidine kinase. It is possible to obtain cells that have mutant forms of one of
these enzymes. Such cells fail to survive in media containing hypoxanthine, aminopterin, and thymidine because of
the blockage of the de novo and salvage nucleotide metabolic pathways. When cells deficient in hypoxanthine
phosphoribosyl transferase are fused with cells lacking
thymidine kinase and the fusion products are placed in
hypoxanthine, aminopterin, and thymidine medium, the
parental cells die but the hybrid cells survive due to intergenic complementation. Other drug resistant mutants that
can be used for selective purposes are adenine phosphoribosyl transferase deficiency (26) and uridine kinase
deficiency (27), among others.
Auxotrophic mutants are another class of markers that
are used extensively for purposes of hybrid selection. The
pioneering work of Puck (28) and Chu (29) resulted in
the isolation and characterization of several auxotrophic
mutants, mainly in Chinese hamster cells. In selection, the
fusion mixture of complementing auxotrophic cell lines is
plated in minimal medium, which allows for growth of
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hybrid cells only.
Another set of conditional lethal mutants that are evolving to be quite useful are those which have temperature
sensitive lesions. Many of the temperature sensitive mutants survive at low temperature (34 °C) and die when
placed at 38.5 °C or higher (for example, see Reference
30). Temperature sensitive cell lines with lesions involving
different genes would complement each other, thus providing a selective system. The temperature selection could
be combined with an auxotrophy or drug selective system.
Recently developed cell separation systems based on
size, light scattering properties (31), and other such determinants might prove to be a useful aid in hybrid selection.
Properties of Rodent-Human Cell Hybrids
Most hybrid combinations result in some degree of
chromosome loss. Intraspecific hybrids lose very few
chromosomes and are generally very stable. Interspecific
hybrids, on the other hand, are characterized by chromosome loss, usually from one of the parental cells. In the
case of rodent-human hybrids the loss is preferentially
human. There is a recent report that is an execption to
this rule (32). The number of human chromosomes retained in such hybrids varies between hybrid cell lines.
The rodent genomic input seems to have an effect on the
extent of chromosome loss. Recently, it was reported that
in Chinese hamster-human hybrids a larger number of
human chromosomes were retained by cells that had two
hamster genomic inputs*. We have made similar observations in mouse-human hybrids. The range of human
chromosome retention, among several hybrids we have
examined, is from 1 to 18.
It is reported that the loss of the human chromosomes
in rodent-human hybrids is random (33, 34). No statistical
tests on the random versus nonrandom nature of chromosome loss have been reported. Norum and Migeon (35)
studied the expression of three human markers at several
times during the life of some hybrid cell lines. They report
that the rate of loss of two autosomal markers are distinct,
indicating nonrandom chromosome segregation. Croce,
Girardi, and Koprowski (36) report that hybrids derived
between SV-40 transformed human fibroblasts and mouse
peritoneal macrophages preferentially retain human chromosome 7 in all the hybrids. We have studied the nature
of chromosome retention in a number of independently
derived mouse-human hybrid cell lines and observed that
chromosome 9 is absent in more cell lines than can be
explained on the basis of random loss. By the same token
chromosome 7 is retained in a larger number of cell lines
than would be expected on a random basis. These results
could be interpreted by postulating a growth inhibitor on
chromosome 9 and a growth promoter on chromosome 7.
Similar nonrandom segregation is reported in Chinese
hamster-human hybrids*.
The mechanism of chromosome loss is not clear. Handmaker (37) studied the segregation and reported that
hybrid cells start with a complete composite genome and
that segregation is initiated at the first division, and that
* MURNAME MJ: A somatic cell genetic analysis of human glycosyl
hydrolases. Ph.D. thesis. Yale University, New Haven, Connecticut, 1975.
the majority of the chromosomes that are lost are lost at
the early divisions. He postulates that there is dominance
of mitotic apparatus in one of the species, resulting in
preferential retention of the genome of that species. Tests
are needed to evaluate his hypothesis.
Cell Genetic Markers
There are several genetic markers that show speciesspecific differences and are thus useful in genetic analysis.
Because most of these are primary or secondary gene
products, the investigators are much closer to the study of
gene action, as opposed to organismic phenotypes, some
of which are several steps removed from the gene.
The most widely used markers are isozymes, multimolecular forms of enzymes. Rodent and human cells
have several enzymes in common, and, although their
biochemical actions are similar, their molecular architecture is sufficiently distinct to distinguish them. The differences are visualized by subjecting cell extracts to starch,
cellogel, agarose, polyacrylamide, or some such electrophoretic system, followed by histochemical staining (38).
Another method to detect specific enzymes is through
incorporation of radioactivity, followed by selective precipitation of product and autoradiography (39). These
methods of detection of enzymes offer an additional advantage. Many of the polymeric enzymes tend to form
heteropolymers composed of rodent and human subunits,
which confirms the hybrid nature of the cell line. It is of
interest to determine whether these hybrid enzyme molecules are more or less effective in the reactions they mediate, as compared with the parental molecules.
Components of cell organelles (for example, mitochondria and ribosomes) offer another set of markers for
study in cell hybrids. Because there is fusion of cytoplasms
as well as nuclei in the formation of hybrids, the retention
of cytoplasmic markers is expected in such hybrids. Mitochondrial DNA and mitochondrial enzymes coded by
nuclear genes have been subjected to extensive analysis.
Segregation or population shifts of human mitochondria
(40), recombination of rodent and human mitochondrial
DNA (41), and location of human nuclear coded mitochondrial enzymes in mouse mitochondria (42) have been
reported. The development of two dimensional electrophoretic systems (43) promises methods for studying
mitochondrial and other cellular proteins in the near
future. Eliceiri (44) studied the fate of mitochondrial
RNA in interspecific cell hybrids.
Another set of primary gene products that might prove
useful is tRNAs. The development of methods for isolation
of specific amino acid acceptor tRNAs promises ways to
study these gene products.
Another set of markers of potential are antigenic in
nature. It is possible to obtain antiserums against human
or rodent cell surface specific antigens that do not cross
react. L»se of such antiserums would permit detection of
antigens coded by specific human chromosomes in normal,
malignant, and differentiated cells and their hybrids. Such
studies have been reported (45, 46) and several others
are under investigation. Similarly, the interactions of
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human and rodent genetic elements in the expression of
specific plasma membrane antigens could be studied*.
Enzymes that might not show electrophoretic differences
can be used as antigens to produce antiserums that can be
used as reagents to detect the presence of specific genes in
hybrid cells. Van Heyningen, Craig, and Bodmer (42)
have reported the detection of the human mitochondrial
citrate synthase in hybrids by the use of such methods.
Others have developed similar antiserums against the
human enzymes isocitrate dehydrogenase (NADP dependent), glucose phosphate isomerase, and hypoxanthine
phosphoribosyl transferase*}-.
The use of auxotrophic and drug resistant markers in
selective systems has been described, and these serve as
excellent markers for study in hybrid cells.
Another class of gene products that are used as markers
are those that are produced by specialized cell types. Some
such markers have been investigated, and these include
albumin and other liver specific functions (47), melanin
production (48), and hemoglobin (49). Such specialized
cell products are useful not only as markers but also in the
study of maintenance of differentiated functions and gene
regulation.
Chromosomes
The development of various staining methods that result
in specific banding patterns makes it possible to identify
individual mammalian metaphase chromosomes. These
methods distinguish between rodent and human chromosomes (for example, see Reference 50) and identify
individual human chromosomes or parts thereof. Recently
developed methods (51,*) enable the detection of relatively small human chromosome segments that might be
incorporated into the mouse genome in mouse-human
hybrids.
Human Gene Mapping
There are three methods of human gene mapping using
somatic cell hybrids. The basic principle underlying all
three methods is the same. Because human chromosomes
are lost as individual units, all the genes that are borne
on any given chromosome would be retained or lost
simultaneously. As such, consistent, concordant segregation
of two or more human markers indicates that the genes
coding for these markers are linked or syntenic. Similar
correlations of expression of specific gene products with
retention or loss of individual human chromosomes shows
the location of the gene with respect to the total chromosome complement.
Gene Mapping Based on Selective Retention or Loss
It is possible, in many cases, to select hybrid cells that
retain or lose a specific human function. For example,
if a mouse cells deficient in thymidine kinase are
hybridized with normal human cells and the hybrids are
selected and maintained in hypoxanthine, aminopterin, and
thymidine medium, the human gene for thymidine kinase
* HECHT T, KURCHERLAPATI R, RUDDLE F: Unpublished results.
t SHIMIZU N, SHIMIZU Y, KUCHERLAPATI R, RUDDLE FH: In preparation.
% FRIEND K, DORMAN B, KUCHERLAPATI R, RUDDLE FH: In preparation.
556
Table 1. Partial Isozyme and Chromosome Data from Four MouseHuman Hybrid Cell Lines*
Cell line
AIM3a
Human enzyme
Peptidase C
Isocitrate dehydrogenase-1
Malate dehydrogenase-1
Lactate dehydrogenase-A
Human chromosome
1
2
3
AIM4a
AIM8a
AIM15a
—
—
+
+
—
+
+
+
—
—
—
+
+
+
+
+
+
—
—
+
+
+
—
+
—
—
—
—
* Concordant segregation of isocitrate dehydrogenase-1 and malate dehydrogenase-1 allows syntenic association of these markers. They can be
assigned to chromosome 2 by correlation of gene expression and chromosome presence. Also note concordance of peptidase C and chromosome 1.
has to be retained for the cells to survive. This means that
the chromosome carrying the gene for thymidine kinase
has to be retained in all hybrids. Weiss and Green (12),
using this rationale in the first series of mouse-human
hybrids, concluded that a C-group chromosome is consistently retained in their hybrids. They concluded that
the gene for thymidine kinase is located on a C-group
chromosome. The advent of more precise chromosome
identification methods showed that the chromosome consistently retained in such hybrids is actually an E-group
chromosome (chromosome 17 or 18) (52). Independent
studies by two other groups (53, 54) showed that the
chromosome is 17. Similarly, selection for human hypoxanthine phosphoribosyl transferase by the hypoxanthine,
aminopterin, and thymidine selective system results in
selective retention of the human X chromosome (33, 34),
making the assignment of the gene for hypoxanthine
phosphoribosyl transferase to the X chromosome. These
are examples of positive selective methods.
In some cases negative selective pressures can be introduced. Pappenheimer and Gill (55) have shown that
mouse and human cells differ in their sensitivity to
diphtheria toxin. Mouse cells are resistant to the toxin,
while human cells are highly sensitive. Examination of
human-mouse hybrid cells showed two classes corresponding to the two parental types of sensitivity. When sensitive
hybrids were treated with diphtheria toxin, a majority of
the cells died. A small fraction of the cells, however,
survived to form colonies. Examination of the chromosome
constitution of these cells before and after diphtheria toxin
treatment showed that the sensitive cells uniformly contained human chromosome 5, while the resistant derivatives have lost that chromosome. The presence or absence
of any other chromosome did not affect the viability of the
cells in the presence of the toxin. From these studies it
was concluded that a genetic element(s) that confers the
diphtheria toxin sensitivity on human cells is located on
chromosome 5 (56).
Some markers such as hypoxanthine phosphoribosyl
transferase and thymidine kinase can be either selected for
or against. Growth of hybrid cells between thymidine
kinase- mouse x thymidine kinase* human cells in bromo-
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Table 2. Hypothetical Chromosome Assignment Panel*
Chromosome
Cell line
A
B
C
1
2
—
—
—
+
—
—
3
4
5
6
7
8
—
+
+
—
—
—
-f
+
—
+
—
+
+
+
+
+
+
—
* When a new marker is tested against this chromosome assignment
panel, if the expression of that market, for example, is H
its gene
can be unequivocally assigned to chromosome 2.
deoxyuridine, a thymidine analogue, selects for the preferential loss of human thymidine kinase. Likewise, 8azaguanine or 6-thioguanine can be used to select against
hypoxanthine phosphoribosyl transferase. The ability to
select for or against these markers makes them extremely
useful in hybrid cell studies. Positive or negative selective
pressures can be applied to genes located on at least six of
the human chromosomes. Considerable efforts are being
made to obtain such markers on all human chromosomes.
An extension of this system is that other markers located
on these selectable chromosomes behave exactly the same
as the selected marker, making it possible to assign their
location on the genome. Thus, genes for phosphoglycerate
kinase, glucose-6-phosphate dehydrogenase, and a-galactosidase are assigned to the human X chromosome (33, 34,
57, 58), and galactokinase to chromosome 17 (59, 60),
to mention a few.
Mapping Based on Concordant Segregation
This method (33, 34, 61) is based on the study and
correlation of a number of phenotypic markers and human
chromosomes in a large series of hybrids. The presence or
absence of a specific marker is tested against all other
markers, and concordant segregation of the markers is
assumed to show synteny. This test is called the synteny
test. The second test consists of correlations of human
gene products with individual human chromosomes, which
shows the chromosomal location of a gene. An example
of this method of analysis is presented in Table 1. This
procedure of gene mapping is the most widely used one
at the present time.
studying as few as 5 appropriate cell lines (62, *, t ) . The
method is based on choosing 5 or more cell lines, among
which the syntactical expression (in terms of chromosome
presence or absence) of each chromosome is unique (see
Table 2). In this method a new phenotype is tested in the
chromosome assignment panel that provides data to assign
a gene to a human chromosome. We have developed some
such assignment panels. These have been tested in our
laboratory with considerable success and are becoming
very useful. We have been able to assign several human
genes by this method. They include hexosaminidase B to
chromosome 5 (63), galactokinase to 17 (59), and uridine
monophosphate kinase to 1*.
One of the most important assumptions in these kinds
of mapping procedures is that a given human gene, if
present in the hybrid cell, would be expressed. As such,
one must be certain that the genes under study do behave
in this manner.
Using the somatic cell hybridization methods, more
than 60 genes have been assigned to 22 human chromosomes. Only chromosomes 22 and Y have no markers assigned to them. Some of the medically significant markers
and their gene locations are presented in Table 3. (For
a more complete list of gene assignments, see References
64 and 65.)
Regional Localization of Human Genes
To develop human cells as favorable genetic material
it is not sufficient to be able to assign genes to chromosomes, but it is necessary to determine their sequence
along the length of the chromosome and to correlate them
with specific cytologic features of the chromosome. Several
methods are available to achieve this goal: [1] use of
human cell lines with known chromosomal aberrations as
parents in hybridization experiments; [2] making use of
spontaneous chromosomal changes that occur in low frequencies in hybrid cells; and [3] inducing chromosomal
* SATLIN A, KUCHERLAPATI RS, RUDDLE FH: Cell genetics. Cytogenetics,
in press.
t RUDDLE FH, KUCHERLAPATI RS: In preparation.
Table 3. Some Medically Significant Markers Assigned by the Somatic Cell Hybridization Methods*
Mapping by Panels
Although this method of parasexual analysis offers
advantages over the usual sexual analysis, it still involves
generation and study of a large number of hybrid cell
lines, each derived from an independent fusion event. An
alternative to this method is to obtain 24 different hybrid
cell lines, each containing a different single human chromosome. Test of this set of cell lines for any new human
phenotypic marker should show the chromosome on which
the gene is located. The generation of such lines has not
been completely successful. Besides, the assignment of a
gene to a chromosome, by this method, relies to a large
degree on negative data, and it would not allow mapping
gene functions that might be dependent on the retention of
more than one chromosome. To alleviate this problem we
have developed a new method called the chromosome assignment panel, that allows reliable gene assignments by
Marker
Chromosome
Assignment
Interferon-1
Galactose-1-phosphate uridyl transferase
Hexosaminidase B
Interferon-2
Diphtheria toxin sensitivity
Human leucocytic antigen
Glutathione reductase
Hexosaminidase A
^-microglobulin
Galactokinase
Poliovirus sensitivity
Adenosine deaminase
Antiviral protein
Xg antigen
Hypoxanthine phosphoribosyltransferase
<z-GalaCtosidase
2
2 or 3
5
5
5
6
8
15
15
17
19
20
21
X
X
X
* For a complete list of gene assignments, see References 65 and 66.
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changes in parental and hybrid cells by physical agents
such as X-rays and biological agents such as certain viruses
that have a propensity to cause specific damages.
The principle associated with all these methods is the
same. If a chromosome could be divided into two or more
constituent parts and if the parts could be reliably identified, it would be possible to correlate gene expression with
parts rather than whole chromosomes. The two types of
aberrations that are useful for this purpose are translocations and deletions. In many cases the results from these
two classes of aberrations, with regard to regional assignment, are indistinguishable.
It is known that the genes for phosphoglycerate kinase,
glucose-6-phosphate dehydrogenase, and hypoxanthine
phosphoribosyl transferase are located on the human X
chromosome. Ricciuti and Ruddle (58, 66) have used a
human cell line having a t (X;14) as the parent in hybridization experiments. In this cell line the breakpoint on the
X chromosome is on the long arm, dividing the chromosome into essentially its constituent long and short arms.
The long arm has been translocated to an almost intact
chromosome 14. At the outset, all the three genes could be
located on the short arm, on the long arm, or distributed
along the total length of the whole chromosome. These
cells were fused with RAG, a hypoxanthine-phosphoribosyl-transferase-deficient mouse cell line. Analysis of a
large series of hybrid cell lines, isolated and maintained in
hypoxanthine, aminopterin, and thymidine medium,
showed that the three markers are retained in all hybrids.
Back selection in 8-azaguanine, which selects for cells that
lack hypoxanthine phosphoribosyl transferase, showed that
all markers are lost simultaneously. From these results it
has been concluded that the three markers are located on
either the long or short arm of the X chromosome. Detailed cytologic anaylsis showed concordant segregation of
the three markers with the long arm. Study of a few spontaneous aberrations showed the order of the genes to be
centromere, phosphoglycerate kinase, hypoxanthine phosphoribosyl transferase, glucose-6-phosphate dehydrogenase.
Studies from other laboratories (see Reference 67) confirmed these conclusions despite a contrary report (68).
Spontaneous aberrations occur with a low frequency
in hybrid cells, and these are found to be useful in gene
localization. Creagan and associates (69), using one such
aberration, tentatively assigned the gene for cytoplasmic
isocitrate dehydrogenase to the long arm of chromosome
2. Studies with other series of hybrids confirmed these
conclusions and provided further localization of this gene
(70).
Study of a spontaneous mouse-human translocation
(50, 54) made it possible to assign the gene for thymidine
kinase to the long arm of chromosome 17. Kucherlapati,
McDougall, and Ruddle (71) showed that the gene is distal to 17q21 by studying a spontaneously arisen t(ll;17).
McDougall, Kucherlapati, and Ruddle (72) have induced
breaks in the long arm of 17 by the use of adenovirus
type 12, and analysis of the resulting cell lines has shown
that thymidine kinase is located in the region 17q21-22.
This assignment was confirmed by independent studies
(73).
558
Similar assignments using other gene markers and artificial chromosomal aberration induction methods have
been reported (74, *).
Some panels to enable regional localization of human
genes are available and others are under construction.
The methods that we have described in this report are
useful for mapping constitutive genes. Genes that are expressed in a few specialized cell types can also be mapped
by somatic cell hybridization methods (see References 7579).
Somatic cell hybridization methods have provided an
alternative to sexual methods of human genetic analysis,
enabling a faster, more reliable, and relatively easier
method of assigning human genes to chromosomes. This
method, in conjunction with other methods, is making
human cells in culture a favorite genetic system for study
of formal genetics, gene action, gene regulation, and the
onset and maintenance of cell differentiation.
* FARRIS L, KUCHERLAPATI R, RUDDLE FH: Unpublished data.
ACKNOWLEDGMENTS: Grant support: in part by NIH grant
GM-09966. Doctor Kucherlapati is an NIH postdoctoral research
fellow.
Received 10 June 1975; accepted 18 July 1975.
• Requests for reprints should be addressed to Raju S. Kucherlapati,
Ph.D., Department of Biochemical Sciences, Princeton University,
Princeton, NJ 08540.
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Health Care in Rural India
In India the people have been given to understand that they are entitled to free medical
care. Now I don't think any government in the world can supply completely free medical care to all the people, because care costs money. Somehow we have to make the
people aware of their needs and also make them contribute to the cost of medical care.
In the villages where we work, we find many people who are willing to spend as much
on a single celebration as they spend on medical care throughout the year. So the money
is there. We need to find ways of inducing communities to take more responsibility for
their own care.
Moreover, existing medical education does not really equip a person to deliver health
care, . . . and we therefore need a different kind of person for this task. Furthermore,
we need people in the rural areas themselves, trained in a very simple way to render
medical care to their own people. . . . I think about 20 000 physicians . . . and perhaps
500 000 people, each placed in a village, who could give simple medical care [would
provide adequate staffing].
In India there are about 120 000 physicians, of whom 20 000 are practising in rural
areas. The problem is how to train and motivate them so that they do not merely
practise curricular medicine but use their training to provide total health care for the
people. We also need to train people in the villages to give simple treatment. Unfortunately, the medical profession is too preoccupied with perfection in medical care:
while insisting on high quality, doctors often forget that at present large numbers of
people are going without any medical care whatsoever. Somehow a transformation
has to take place within the medical profession so as to help the government train
people to provide simple care in the villages.
R. S. AROLE
Health Care for Rural Communities
WHO Chronicle 29:257-263, 1975
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