The murine homologs of two viral oncogenes associated with tyrosine-specific kinase activity have been assigned to different loci in the mouse genome. The segregation of restriction site polymorphisms, as detected by probes that are specific for endogenous c-fes and c-src sequences, was followed in the DNA of recombinant inbred strains. The c-fes gene was mapped to the proximal portion of chromosome 7, very close to the Gpi-1 locus, whereas c-src was linked to the Psp locus on the distal half of chromosome 2

Blatt, Cila

Franchini, Genoveffa

Harper, Mary E.

Nesbitt, Muriel N.

Simon, Melvin I.

Caltech Authors - Main

Vol. 4, No. 5MOLECULAR AND CELLULAR BIOLOGY. May 1984, p. 978-981
0270-7306/84/050978-04$02.00/0
Copyright (C 1984, American Society for Microbiology
Chromosomal Mapping of Murine c-fes and c-src Genes
CILA BLATT,'* MARY E. HARPER,' GENOVEFFA FRANCHINI,2 MURIEL N. NESBITT,3 AND MELVIN I.
SIMON'
The Agouron Institute, La Jolla, California 920371; Laboratory of Tumor Cell Biology, National Cancer Institute,
Bethesda, Maryland 202052; and Department of Biology, University of California, San Diego, La Jolla, California 920933
Received 27 October 1983/Accepted 2 January 1984
The murine homologs of two viral oncogenes associated with tyrosine-specific kinase activity have been
assigned to different loci in the mouse genome. The segregation of restriction site polymorphisms, as
detected by probes that are specific for endogenous c-fes and c-src sequences, was followed in the DNA of
recombinant inbred strains. The c-fes gene was mapped to the proximal portion of chromosome 7, very
close to the Gpi-J locus, whereas c-src was linked to the Psp locus on the distal half of chromosome 2.
Oncogenic retroviruses contain transforming genes (v-onc
genes) as an integral part of their genomes. It is believed that
these viruses are a result of genetic recombination between
retroviral sequences and distinct cellular sequences (c-onc
genes), which are responsible for their acute transforming
ability. The products encoded by some identified v-onc
genes are associated with tyrosine-specific kinase activity
(src, yes, abl, ros, andfes; reviewed in reference 2). Nucleo-
tide sequence analysis (9, 26) and measurements of immuno-
logical cross-reactivity (1) support the idea that some of
these oncogenes (the src family) are evolutionarily related.
The recent discovery of a gene that is homologous to v-src
and v-abl in Drosophila melanogaster DNA further suggests
that these oncogenes may have evolved from a common
ancestral gene (14). It is of interest, therefore, to determine
the relative locations of the cellular oncogenes in the mam-
malian genome and to find out whether members of the src
family of genes are clustered on the same chromosome.
Furthermore, the importance of the chromosomal locations
of cellular transforming genes has been underscored by the
demonstration that specific chromosomal translocations in-
volving sites adjacent to specific c-onc genes are associated
with murine and human lymphomas (4, 16, 23, 29). While
this manuscript was still being prepared, c-src and c-fes were
assigned to chromosomes 2 and 7, respectively, by the use of
mouse-hamster somatic cell hybrids (A. Sakuguchi, personal
communication) (18). We have mapped the mouse cellular
homologs of v-fes and v-src to determine their relative
chromosomal locations and to examine the possibility of
their involvement in DNA rearrangements in the neoplastic
transformation.
The regional chromosomal locations of c-fes and c-src
were studied by following the segregation of restriction
fragment length polymorphisms involving these genes within
the DNA of two sets of recombinant inbred (RI) strains.
These strains were derived from crosses between inbred
strains A/J (A) and C57BL/6J (B) or from crosses between
strains B and DBA/2J (D), which resulted in AxB and BxA
RI strains (22) and B xD RI strains (Jackson Laboratory, Bar
Harbor, Me.; 30, 31), respectively.
Gene mapping of RI strains is accomplished in three steps.
(i) Parental strains are screened for phenotypic or genotypic
variation, in this case by using a nucleic acid probe (24) that
* Corresponding author.
detects a specific restriction site polymorphism in the DNA.
(ii) The hybridization pattern of each of the RI strains is
determined, and the resemblance to one parental strain or
the other is ascertained, resulting in a strain distribution
pattern (SDP) for a given genetic locus (see Table 1). (iii) The
newly determined SDP is compared with SDPs of previously
mapped genes to ascertain the degree of linkage and, thus,
the chromosomal position of the particular gene (8, 30).
Polymorphism within the murine c-fes locus was detected
by Southern blotting of restriction endonuclease-digested
DNA of the two progenitor strains A and B, followed by
hybridization with a c-fes probe (7). The pattern obtained by
using DNA digested with four different restriction enzymes
suggests the existence of a single locus containing the murine
c-fes gene (Fig. 1). Except for EcoRI, no other restriction
enzymes used (HindIII, PstI, and BglII in Fig. 1 and MspI,
PvuII, and XbaI, not shown) generated a polymorphic
restriction pattern among A, B, and D mice. The EcoRI
fragments that hybridize to c-fes differ in size: a 13-kilobase
fragment is present in the A strain, and a 12-kilobase
fragment is present in the B strain. The polymorphism in the
size of the EcoRI fragments was used to follow the segrega-
tion of the variants in the Ax B and B xA RI strains. A total
of 30 RI strains were tested; two representative Southern
blots are shown in Fig. 2. That RI strains show both parental
phenotypes is due to heterozygosis; i.e., the c-fes alleles
were still segregating, and these RIs could not be used in the
analysis. The results showed that 23 of the 30 RI strains were
homozygous with respect to the size of the EcoRI fragment
containing the c-fes gene. Comparison of the SDP for c-fes
with SDPs of other genes indicated tight linkage between c-
fes and genes located on chromosome 7 (Table 1). Thus, c-
fes is located between Gpi-i (glucose phosphate isomerase-
1) (15) and Tam-i (tosyl arginine methylesterase-1) (27), with
a distance of 2.6 + 2 centimorgans from Gpi-J and 6 + 4
centimorgans from Tam-i.
A polymorphic pattern involving the murine c-src locus
was obtained by using DNA of parental strains B and D,
digested with HindIII and hybridized with a v-src probe (6).
Other restriction enzymes, such as EcoRI, PstI, and BglII,
generated identical patterns among the mouse strains tested.
The v-src probe hybridizes to a single 13-kilobase DNA
fragment in D mice and hybridizes with two DNA fragments
of 16.5 and 14 kilobases in the DNA of B mice. The
segregation of these alleles was followed in 26 B xD RI mice,
and the SDP was determined (Table 2). Analysis of SDPs
indicated that the murine c-src gene is located on chromo-
978
NOTES 979
TABLE 1. SDP of c-fes and neighboring genes on chromosome 7
SDP of:
Locus Ax B RI strain BxA RI strain
1 2 3 4 5 6 9 10 12 14 15 20 21 25 1 3 4 6 7 13 14 19 23
c-fes B A A B A A A B A A A B A B B B A A A A A A A
Gpi-la B A A B A A A B A A A B A B B B B A A A _b B A
Tam-lc B B A B A A A B A B A B A b B B A A B A A B A
cd B A A B B A A B A A B B B B B B A B A A A A B
Hbbe B A A B A A A B A A B B B B B B A B A A A A A
a Glucose phosphate isomerase-1 (15).
b_, Not tested.
Tosyl arginine methylesterase-1 (27).
d Coat color (albino).
e Hemoglobin chain (32).
some 2 by linkage (2.2 + 1.6 centimorgans) to Psp (parotid
secretory protein) (13), which resides on the distal half of the
chromosome. However, the orientation of Psp and c-src
with respect to the centromere was not posssible because of
a lack of SDPs for other genetic markers on this portion of
chromosome 2. This result places c-src more than 20 centi-
morgans away from the c-abl gene, another member of the
tyrosine kinase genes that maps very near to the P2-microglo-
bulin gene (21). It has been suggested that the genesis of
myeloid leukemia is greatly influenced by genetic informa-
tion of chromosome 2 since partial deletions of chromosome
2 have been associated with 49 of 52 cases of the disease
Pst I HindM
..
a: <
,Y0 U
< C: CD
Eco RI
r- z ----
=N c-s --
c: < 4 I.-
< <~n an a, -
am m "12-0
-7.0
(11). Further studies are necessary to examine the involve-
ment of c-src and c-abl on chromosome 2 with leukemia.
The mouse c-fes gene maps to the proximal portion of
chromosome 7 between the Gpi-J and Tam-i genes. It is
interesting that although chromosome 7 has not been shown
thus far to be involved in specific translocations in malignan-
cies, a number of genes potentially involved in oncogenesis
have been mapped to chromosome 7. These include the
ecotropic murine leukemia virus loci Akv-J ofAKR mice and
Fgv-J of C3H/Fg mice (17), two endogenous mink cell focus-
inducing viral sequences (2a), Mtv-J (a mammary tumor viral
sequence [31]), and the c-Ha-ras-1 gene (18).
Studies with human-mouse somatic cell hybrids assigned
the human c-fes gene to chromosome 15 (5, 12), and more
recently it has been sublocalized to 15q25-26 by in situ
hybridization (10). Comparative mapping suggests that a
portion of mouse chromosome 7 is homologous to a region of
human chromosome 15, since the mitochondrial isocitrate
A x B RI STRAINS
d3 I4 2 5 1//
3 3 4 21 5 5 6 6 7 7 8 8 9 9 10 11
-/44
_ _ _ _ _ g _ _
20 /0 $/ s // /20 20 18 18 17 {5 15 14 14 12 12
FIG. 1. Detection of c-fes sequences in the DNA of four inbred
mouse strains. High-molecular-weight liver DNA was digested with
endonuclease restriction enzymes (4 U/,ug), followed by electropho-
resis and Southern transfer (28). The blot was hybridized with a 3-P-
labeled c-fes probe, recombinant plasmid pN26 (7). Spleen (S) and
liver (L) DNAs of D mice digested with EcoRl exhibit an identical
hybridization pattern. The variation between DNA of A and B mice
was used to determine the segregation of the c-fes gene in the Ax B
RI strains. Numbers on the right indicate fragment size in kilobases.
FIG. 2. Segregation of the allelic EcoRI restriction fragments in
AxB RI strains as shown by hybridization with the c-fes probe.
Strains heterozygous at the c-fes locus (AxB 7, AxB 8, AxB 17,
and AxB 18) were not included in the SDP.
Bg IlE
.-
-.z£r 4 c-
~d it
4c 0) L
-,
-3 c..J
-j
-
_ CC)
r P-
x OD V)
cK < a c;
do a" d
-2.2
1.6
VOL. 4, 1984
MOL. CELL. BIOL.I . ,,,, , v
) > #\ s
t->I ) i) [s 1.) {)
.1n,. 1) I) is I.) 14 i:;
[ }. 1, } t t1 s , < , k } ]' '\ [) 1't) 1 4 1' ;
I) I) I) BS B I) 1) HS IJ I) BS
I_.I
i*) 1) 1)
i) 1) 1)
-
I'i ;
) l'1 i ) )
1) B1 1) 1)
BXD RI STRAINS
8 9 12 ' i5 6 _ 2O 21
ag .-If
.
2 22 23 24 25 27 28 29 30 3.i 32 B D
t6
_ _
qbA am
FIG. 3. Segregation of the allelic HindIII restriction fragments in
BxD RI strains as shown by hybridization with the v-src probe (6).
dehydrogenase locus (Idh-2) is located on mouse chromo-
some 7 (19) and on 15q21-qter in humans (20). This finding is
of interest since the distal half of human chromosome 15 is
frequently involved in a specific translocation in acute
promyelocytic leukemia (25). It would be of interest to
determine whether the murine c-fes gene and a region of
chromosome 7 are involved in similar rearrangements in
malignant diseases in mice.
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VOL. 4, 1984


Chromosomal mapping of murine c-fes and c-src genes

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Chromosomal mapping of murine c-fes and c-src genes

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