Download regional mapping of the gene coding

J. Cell Set. 53, S45-254 (198a)
Printed in Great Britain © Company of Biologists Limited 1982
245
REGIONAL MAPPING OF THE GENE CODING
FOR ENOLASE-2 ON HUMAN CHROMOSOME 12
MARTHA LIAO LAW AND FA-TEN KAO
Eleanor Roosevelt Institute for Cancer Research and The Department of Biochemistry,
Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Col.
80262, U.S.A.
SUMMARY
Enolase-2 (ENO2), previously termed 14-3-2 protein, is an isozyme of enolase that is
enriched in neuronal tissue. The gene coding for ENO2 was previously assigned to human
chromosome 12. The present study presents data for a regional mapping of gene ENO2 using
cell hybrids containing various deletions of human chromosome 12. These deletions were
produced by treatment with chromosome-breaking agents. Cytogenetic analysis has allowed
assignment of ENO2 to the short arm of chromosome 12, in the region of pter-pi2O5. This
assignment is consistent with the segregation pattern of the 93 hybrid clones analysed. The
segregation pattern has also established the linear order of 6 genes on chromosome 12:
pter - TPI- GAPD - LDHB - ENO2 - centromere - SHMT - PEPB - qter. Estimation of
the relative distances between the 6 genes on chromosome 12 has been made by a statistical
mapping analysis of the segregation data of the hybrid clones. A set of deletion hybrids containing various combinations of these 6 markers has been established for a rapid regional
mapping of genes in one of these regions on chromosome 12.
INTRODUCTION
Previously we reported a regional assignment of 5 genes on human chromosome 12
(Law & Kao, 1979). These genes code for the following enzymes: triose phosphate
isomerase-i (TPIi), glyceraldehyde-3-phosphate dehydrogenase (GAPD), lactate
dehydrogenase B (LDHB), serine hydroxymethyltransferase (SHMT), and peptidase
B (PEPB). The linear order and the map positions of these genes have been determined
by both cytogenetic and statistical mapping analyses. In this paper, we present data
for a-regional assignment of another gene on human chromosome 12, coding for
enolase-2 (ENO2; EC 4.2.1.11), an enzyme previously termed neuronal-specific
14-3-2 protein. The chromosomal assignment of the ENO2 gene was made by
Grzeschik (1975) and Herbschleb-Voogt et al. (1978). An abstract reporting preliminary studies of this regional assignment has been presented (Law & Kao, 1980).
MATERIALS AND METHODS
Cells and cell culture
The various hybrid cells used in this study were derived from fusions between human cells
and the glycine-requiring mutant gly'A of the Chinese hamster ovary (CHO-K1) cells. The
chromosomal deletions induced in human chromosome 12 were achieved by treatment of the
cells with either X-rays or 5-bromodeoxyuridine +near-visible light, as described in detail
246
M. L. Law and F.-T. Kao
previously (Law & Kao, 1978, 1979). The hybrid clone 12A containing theglyh genome and a
single human chromosome, 12, was grown in glycine-free medium F12D supplemented with
10% of the macromolecular fraction of foetal calf serum. The CHO-K1 cells and the gly'A
mutant were grown in complete F12 medium supplemented with 8% foetal calf serum.
The following cells were used to assay for enolase-2 activities: IMR-32, a human neuroblastoma cell line obtained from the American Type Culture Collection; P2, a mouse
neuroblastoma cell line obtained from K. Spuhler; FS10, a diploid human fibroblastic
culture established from a foreskin biopsy. These cells were grown in complete Fi2 medium
supplemented with 8 % foetal calf serurn. In addition, the enolase-2 activity has been assayed in
various tissue cells obtained from human biopsy, human brain grey and white matter, and
Chinese hamster brain tissue biopsy. These tissue cells were used directly for enzyme assays
without in vitro culture.
Cellogel assay for enolase-z
The crude cell extracts were prepared from the cultured cells and cell hybrids using the
procedures previously described (Law & Kao, 1978). Similar methods were used for cells from
tissue biopsies. Horizontal Cellogel electrophoresis (Meera Khan, 1971) was carried out at
room temperature using c o i M-phosphate buffer (pH 6-2), for detecting enolase activity. The
staining solution of Chen & Giblett (1976) was used, which contained c i M-Tris-HCl buffer
(pH 7-8), 8 mM-MgSO4, o-i M-KCI, I mM-2-phosphoglycerate, 2 mM-ADP, 1 mM-NADH,
10 units per ml of pyruvate kinase, 30 units per ml of lactate dehydrogenase. After staining, the
dark enolase bands can be seen against a fluorescent background, by long-wave ultraviolet
illumination. Since ENO2 is generally less active than enolase-i (ENOi), it is necessary to
stain longer for detecting ENO2 activity. In order to avoid over-staining of ENOi in some gels,
the reaction was carried out first for 30 min; the gel was then cut and ENOi bands were
removed. The gel was stained for another 90 min to reveal ENO2 bands.
Cytogenetic analysis
The trypsin-banding techniques described previously for karyotypic analysis were used (Kao,
Jones & Puck, 1976).
RESULTS
Enolase-z activities in various tissues and cultured cells
Fig. i A shows the enolase patterns of the various neuronal cells. Enolase is a dimer
consisting of a and /? subunits. In Cellogel assays, 3 bands are formed: the cathodal
band aa, the anodal band /?/?, and the middle band aft. The human homodimer a ^ is
termed human enolase-1; the human homodimer Pxft\ is termed human enolase-2;
the middle band is a heterodimer ct1fiv Chen & Giblett (1976) have shown that ENO2
is abundant in human brain tissue cells, but this isozyme is also present in lower
concentrations in other tissues and in cultured skin fibroblasts.
In Fig. 1 A, lane 1 shows a mouse neuroblastoma cell line P2, which contains 2
bands, presumably a homodimer oioux type (lower band) and a heterodimer ay? (upper
band). Lanes 2, 4 and 5 show tissue cells obtained from, respectively, human brain,
human grey matter, and human white matter. Each of these cell types has 3 bands
corresponding to (from the bottom): a ^ , cLxfSx and fixP\- Lane 3 shows a human
neuroblastoma cell line IMR-32. The homodimer fix^ is weak, but shows a definite
band after 2 h of staining.
Regional mapping of human enolase-z
247
Enolase-z assay in hybrid cells
In Fig. IB, lane 1 shows the enolase assay in the hybrid clone 12A, which contains
a single human chromosome 12. The cathodal band in lane 1 is the homodimer a^tz
in which the a 2 subunit is coded for by a CHO gene. The band migrated to the same
position as the cathodal band of CHO-Kl (lane 2). After 2 h of staining, a second band
appeared above the a^xt band in 12A (lane 1), but not in CHO-Kl (lane 2). This bard
is interpreted to be a heterodimer composed of subunits at^1 in which the fix subunit
Fig. 1. Cellogel electrophoretic patterns stained for enolases. Lanes in A: I, mouse
neuroblastoma cell line P2; 2, human brain tissue cells; 3, human neuroblastoma cell
line IMR-32; 4, human brain grey matter; 5, human brain white matter. Lanes in
B: 1, 12A; 2, CHO-Kl; 3, Chinese hamster brain tissue cells; 4, human brain tissue
cells; 5, cultured human fibroblasts FS10. The human enolase subunits are designated
as a t and filt and the Chinese hamster enolase subunits are designated as otj and j3t.
The band a-ifii in B, lane 1, was very faint after staining for 45 min, but became darker
after longer staining. The gels in A and B were stained for 2 h. The subunit composition of each enolase band is indicated by arrows.
is coded for by a gene on human chromosome 12. The band a ^ migrated slightly
faster than the Chinese hamster heterodimer ai^l (second band from top in lane 3),
and considerably faster than the human heterodimer a2y32 (lanes 4, 5). The presence
of the band a ^ in the 93 hybrid clones assayed has been used as indication of the
presence of the ^ subunit of the ENO2 marker. In each individual assay for ENO2
activity in the hybrid clones, cell extracts from CHO-Kl and 12A were always
included as controls and, in every case, the band a2/?! was absent in CHO-KI and
present in 12A.
Grzeschik (1975) showed the formation of interspecies heteropolymers between
human and mouse enolase subunits. We demonstrated here that similar heteropolymers can also be formed between human and Chinese hamster subunits.
248
M. L. Law and F.-T. Kao
The ENO2 activity and the activities of the 4 other isozymes were assayed in 93
hybrid clones and the results are presented in Table 1. Of these 93 clones, 44 were
isolated by method B (Law & Kao, 1978), in which human cells were treated with
chromosome-breaking agents followed by fusion with the gly~A mutant. SHMT+
hybrids were selected in F12D for the retention of human chromosome 12, either
intact or partial. The other 49 hybrids were isolated either by method A (Law & Kao,
1978), or by a modified method A (Law & Kao, 1979), in which the 12A cells were
treated with chromosome-breaking agents and the survivors were isolated.
Table 1. Isozyme analysis of 93 hybrid clones each possessing at least one human marker
on chromosome 12
Human isozyme marker
—
TPI
GAPD
+
+
+
—
+
-
+
+
+
+
+
+
+
+
+
+
—
—
—
—
—
—
—
+
—
—
+
+
+
+
+
—
+
—
Method B +
—
+
—
—
—
—
-
Method A
+
—
LDHB
-
-
-
+
—
ENO2
-
+
—
PEPB
Number of
hybrids
+
+
18
+
+
+
+
+
4
3
4
+
+
+
+
+
+
+
+
—
+
—
—
+
-
1
2
i
1
1
1
8
+
+
+
+
44
2
SHMT
The 93 hybrid clones can be grouped into various phenotypic classes and arranged
in order according to the map position of the genes on the chromosome. From the
segregation patterns shown in Table 1, it is logical to place ENO2 between LDHB and
SHMT. Moreover, ENO2 was assayed and shown to be positive in the 2 previously
described independent clones MAi and MA2, which had large deletions in the long
arm of chromosome 12 with the breakpoint at qi2 (Law & Kao, 1979). Thus, based on
the karyotype and the phenotype (TPI+GAPD+LDHB+EN^+SHMT-PEPB-) of
these 2 clones, ENO2 can be placed in the region pter-qi2, or between LDHB and qi2.
Cytogenetic analysis
The hybrid clone A9 was derived from 12A after treatment with bromodeoxyuridine
+ near-visible light (Law & Kao, 1979). The phenotype of A9 is TPI-GAPD"LDHB-ENO2-SHMT+PEPB+. This clone has a terminal deletion in the short arm
with the breakpoint at pi205 (Fig. 2). Thus, like TPI, GAPD and LDHB, ENO2 can
also be assigned to the region pter-pi2O5. This regional assignment is consistent
with the location of ENO2 being pter-qi2 as derived from clones MAi and MA2.
Regional mapping of human enolase-2
249
Statistical mapping analysis
Regional mapping by statistical analysis of the segregation data (Table 1) was
performed following closely the procedures described in our previous paper (Law &
Kao, 1979). We used the methods of Goss & Harris (1975, 1977a, b) to derive the
linear order of the 6 genes on chromosome 12 and to estimate the relative distances of
these genes in relation to the selected locus SHMT.
B
D
I
Fig. 2. Trypsin-banded metaphase chromosomes in the 12A cell (A) containing a single
human chromosome 12 (Hui2, arrow), and the A9 cell (B) containing a partial human
chromosome 12 with a terminal deletion pter-pi2os (arrow). In the lower section are
presented the enlarged human chromosome 12 (c) and the partial chromosome 12 (D).
The segregation data of the 44 deletion hybrids (Table 1, method B) show that TPI
is lost in 21 hybrids (48 ±7%); GAPD is lost in 16 hybrids (36 ±7%); LDHB is lost
in 14 hybrids (32 ±7%); ENO2 is lost in 9 hybrids (20 ±6%); and PEPB is lost in 15
hybrids (34 ± 7%). The numbers in brackets refer to the frequency ± standard error.
These segregation frequencies reflect the relative distances between SHMT and
any one of these markers. Thus the following order of relative distances can be arranged, beginning with the smallest distance: ENO2, LDHB, PEPB, GAPD, TPI. This
order of relative distances is consistent with our previous results (Law & Kao, 1979),
and also adds the ENO2 marker, which is closest to the locus of selection.
Our previous data based on statistical calculation and cytogenetic analysis have
also shown that TPI, GAPD and LDHB are on the short arm of chromosome 12,
9
CEL53
M. L. LawandF.-T. Kao
250
while SHMT and PEPB are on the long arm. It is evident from the segregation patterns
that, when ENO2 is lost, TPI, GAPD and LDHB are also lost. However, the reverse
is not true; that is, when TPI, GAPD or LDHB is lost, there is on average a 50%
chance that ENO2 is still retained in the hybrid. Therefore, we conclude that ENO2
is closer to SHMT than the other markers. PEPB appears to segregate independently
of ENO2, indicating that they are on opposite sides of the selected locus SHMT.
Indeed, our cytogenetic data support this conclusion.
Table 2. Estimation of target size {i.e. distance between 2 loci) using co-transfer frequency
(F) of unselected markers with SHMT
Unselected marker
TPI
No. of clones exhibiting unselected
markers and SHMT
23
F
-log F
-logF'
GAPD
LDHB
ENO2
PEPB
28
3°
35
29
066
0-18
0-52
0-28
0-64
0-19
068
080
0-17
I'OO
o-68
061
o-io
0-36
0-64
Total number of clones = 44
•SHMT
s
L90
SHMT
•TPI
-GAPD
•LDHB
•ENO2
CO
0-6
TPI
GAPD
LDHB
EN02
6
-
CO
•PEPB
6
PEPB
Fig. 3. Diagrams showing cytogenetic map (left) and statistical gene map (right) of
human chromosome 12.
Finally, the relative distance between 2 loci can be estimated using a simple target
theory (Goss & Harris, 1975, 1977a, b), which to a first approximation equates the
relative distance between 2 markers to — log of the frequency of hybrids possessing
both markers. Table 2 presents the calculations based on these analyses. The last row
represents the relative distance between SHMT and each of the syntenic markers,
normalized to a distance of i-oo between TPI and SHMT. Corrections for interstitial
deletions and other complex chromosomal rearrangements as described by Goss and
Harris were not attempted here because of the small sample size of our deletion
hybrids. Thus, the relative distance presented here is only an estimate.
Regional mapping of human enolase-2
251
Comparison of cytogenetic and statistical maps of human chromosome 12
Fig. 3 presents the gene maps of human chromosome 12 constructed by both
cytogenetic and statistical analyses. In general, the order of the 6 markers on chromosome 12 is consistent in the 2 maps. A significant difference in the 2 maps exists in the
relative positions of the 4 genes on the distal part of the short arm. The cytogenetic
map assigns the 4 genes to the region pter-pi2O5, but the statistical map shows that
the 4 genes occupy a larger section, with ENO2 extended to the band pi 1.
DISCUSSION
Enolase, or 2-phospho-D-glycerate hydrolase, reversibly converts 2-phosphoglycerate to phosphoenol pyruvate in the glycolytic pathway. Three enolase isozymes
have been identified: a non-neuronal enolase (ENOi), a neuronal enolase (ENO2),
and a hybrid form of ENOi and ENO2 (Schmechel et al. 1978). ENOi is present in
all tissues and consists of 2 identical subunits ct^x.lt each of about 39000 molecular
weight. ENO2 is enriched in neuronal tissue but is also present in smaller quantities in
other tissues. This enzyme consists of 2 identical subunits filfi1 each of about 43 500
molecular weight. The third form of enolase is a heterodimer consisting of cc^i
subunits.
The locus for human a subunit ( a j has been mapped to chromosome 1 both by
family study (Giblett, Chen, Anderson & Lewis, 1974) and by cell hybrid analysis
(Meera Khan, Deppert, Hagemeijer & Westveld, 1974). The locus for human fi subunit (/fj) has been mapped to chromosome 12 using cell hybrids (Grzeschik, 1975;
Herbschleb-Voogt et al. 1978). Here we present data to assign regionally the fix locus
for the enolase-2 phenotype to the short arm of chromosome 12, in the distal region
pter-pi2O5. This regional assignment is consistent with the segregation data of 6
syntenic genes on human chromosome 12 (Table 1). The cytogenetic and segregation
data also place ENOz between LDHB and the breakpoint pi205. Previously, TPI
and GAPD were regionally assigned to pi3 by Serville et al. (1978), and LDHB to
I2-I-I2-2 by Rethore et al. (1975). In their nomenclature, the band pi2 is divided
into 3 equal parts designated 12-1, pi2-2 and 12-3, with 12-1 being contiguous with
pi 1. Thus, the region pi2-i-pi2-2 is roughly equivalent to the region 1201-1207 in
the system recommended by the Paris Conference Supplement (1976). Based on this
regional mapping of LDHB and our assignment of ENOz between LDHB and pi205,
we can further localize both LDHB and ENO2 to P1207-P1205, with the order of the 2
genes being LDHB distal and ENO2 proximal to the centromere.
In the IV International Workshop on Human Gene Mapping, Bruns & Regina
reported a possible second TPI locus (TPI2) on human chromosome 12 coding for a
heat-labile subunit of the TPI isozyme. In our assays for this heat-labile TPI2
isozyme, we found no segregation between TPIi and TPI2 in the 93 hybrids analysed,
as we reported previously for a smaller number of these hybrids (Law & Kao, 1980).
In the present paper, we used the term TPI to represent both of these isozymes; the
9-2
252
M. L. Law and F.-T. Kao
precise nature and the identity of TPI2 require further biochemical and genetic
studies.
The set of hybrids carrying various marker deletions can divide chromosome 12 into
the following 8 regions: pter -1- - TPI -2- - GAPD -3- - LDHB -4- - ENO2 -8- centromere -6- - SHMT -7- - PEPB -8- - qter. Thus a set of a minimum of 5 clones
exhibiting unique combinations of these markers can be selected from Table 1 and
used for rapid regional mapping of other genes assigned to chromosome 12. However,
the assignment to regions 5 and 6 will also require karyotypic analysis. A similar set of
deletion hybrids has been established for the X chromosome by Becker et al. (1979).
We previously used the statistical mapping analysis of Goss & Harris (1975, 1977 a,b)
to estimate distances for the 5 genes on human chromosome 12 (Law & Kao, 1979).
The statistical map in general agrees with the cytogenetic map. However, Serville
et al. (1978) assigned TPI and GAPD to pi3 by cytogenetic analysis, while our
statistical map places GAPD in pi2. Moreover, the statistical map position for ENO2
is more proximal to the centromere than its position in the cytogenetic map (Fig. 3).
By comparing the 2 maps for the 4 genes on the short arm, it appears that the genes on
the statistical map are extended to a greater distance. The significance of this disparity
requires further study.
It is worthwhile to point out that in the statistical mapping of the human chromosome 1, Goss & Harris (19776) found a similar non-coincidence, one interpretation
of which was that radiation-induced rearrangements occurred preferentially in
Giemsa-stained light material.
It should also be pointed out that the cytogenetic map is constructed on the basis
of the location of genes in the highly condensed metaphase chromosomes, whereas the
statistical map is based on the location of genes in the extended state of the interphase
chromosome in which radiation-induced breaks occur. Thus, while the statistical
map measures the distance between genes in the extended DNA sequences, the cytogenetic map measures the distance between genes in the condensed metaphase
chromosomes. Since meiotic crossing-over takes place during close-pairing of
homologous chromosomes in an extended state, the statistical map should resemble
more closely the genetic map based on recombination events observed in higher
organisms.
The regional assignment of ENO2 to the short arm of chromosome 12 is particularly
interesting. It is the fourth enzyme of the glycolytic pathway for which the gene has
been assigned not only to the same chromosome, but also to the same arm. Since these
4 genes are all separated by some distances as shown in the map, they are clearly not
contiguous in the DNA sequences. However, the assignment of 4 genes related in a
common pathway to a specific region of the chromosome may have some significance
in evolution and possibly in gene regulation. The possible relationship between the
coordinate regulation of functionally related genes and the physical linkage of these
genes on a segment of the chromosome certainly requires further investigation.
Mapping of these 4 genes in other species may provide additional insight into the
significance of this linkage relationship.
Regional mapping of human enolase-2
253
This investigation is a contribution from the Eleanor Roosevelt Institute for Cancer Research
and the Florence R. Sabin Laboratories for Development Medicine (Contribution no. 350),
and the Department of Biochemistry, Biophysics and Genetics, University of Colorado Health
Sciences Center, Denver, Colorado. This work was supported by grants from the American
Cancer Society (CD105) and the National Institutes of Health (GM26631, HD02080). We
thank Drs T . T . Puck and D. Patterson for critical reading of the manuscript. This paper is
no. 34 in the series entitled 'Genetics of Somatic Mammalian Cells'. The preceding paper is
by Meisler, Wanner, Kao & Jones (1981).
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{Received 2 June 1981)