LARRY J. SHAPIRO§, JIM SCHRODERO, AND DAVID KORN*t. *Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 5270-5274, August 1985 Biochemistry
Assignment of the gene for human DNA polymerase X chromosome
a
to the
(monoclonal antibodies/gene mapping)
TERESA SHU-FONG WANG*t, BARBARA E. PEARSON*, HELi A. SUOMALAINENt, T. MOHANDAS§, LARRY J. SHAPIRO§, JIM SCHRODERO, AND DAVID KORN*t *Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305; tFolkhalsan Institute of Genetics, Helsinki, Finland; and Division of Medical Genetics, Department of Pediatrics, Harbor-University of California at Los Angeles, Torrance, CA 90509
Communicated by I. Robert Lehman, April 11, 1985
(2bR-2) reactivation of the human HPRT locus; they express human HPRT, but not human glucose-6-phosphate dehydrogenase (G6PD) or a-galactosidase (10, 11). Hybrid clone XVIII-54A-2a was derived from the fusion of Chinese hamster V79/380-6 and lymphoblasts (GM 7773) isolated from a female patient with a de novo interstitial deletion of most of band Xp2l [46,X,del(X)(pter--21.3: :p21. 1-qter)] associated with random X inactivation (12). In this hybrid the X with the deletion is the active one, and the inactive, structurally normal X is not present. All hybrid clones were maintained and propagated as described in their original references. Biochemical and Immunochemical Assays. The growth and extraction of human or rodent cells, the preparation and assay of DNA polymerase a fraction IIA, the binding assays of antibodies with polymerase a fractions, and the properties of monoclonal antibodies SJK 132-20 and SJK 237-71 and monoclonal nonimmune immunoglobulin, P3 IgG, have been described (1, 2). To measure X chromosome marker enzymes, extracts (2) of hybrid clones were assayed for G6PD (13) and HPRT (14) activities. Solid-phase radioimmunoassay of human DNA polymerase a antigen was performed by coating polyvinylchloride microtiter plates with SJK 132-20 IgG, which binds both mouse and human polymerase a (Fig. lA), adding a fixed quantity of polymerase a activity from cell extracts of interest, and probing for the presence of the human antigen with 1251-labeled SJK 237-71 IgG, which discriminates the human and rodent species (Fig. 1B). Under the conditions of the assay, murine polymerase a resulted in 178 ± 88 cpm of bound 1251 (mean ± 2 SD, n = 10). Samples with < 266 cpm were defined as negative, and samples with 2 532 cpm, as positive, for human polymerase a antigen. Chromosomal Analysis. Karyotyping was performed simultaneously with each experiment by the G-11 (15) or modified trypsin-Giemsa (16) banding method. In each case 20 mitotic cells were analyzed in detail and photographed. Counterselection of Hybrid Clones Containing Human X Chromosomes. Hybrid cells were weaned from hypoxanthine/aminopterin/thymidine (HAT) medium into Dulbecco's modified Eagle's medium (DMEM) and were then grown in DMEM with 60 ILM 6-thioguanine (6TG medium) (17) for several passages. The populations that survived the selection were maintained in 6TG medium.
We have applied an assay based on a monoABSTRACT clonal antibody that discriminates the activity of human DNA polymerase a in rodent-human somatic cell hybrid clones to identify a single genetic locus that is both necessary and sufficient for the expression of DNA polymerase a. We have mapped this locus to the short arm of the human X chromosome, near the junction of bands Xp21.3 and Xp22.1, and demonstrated that it is not expressed from an inactive X chromosome.
We have described (1) a panel of monoclonal antibodies specific for DNA polymerase a and have used them for immunoaffinity purification of a complex, presumptively native, form of the enzyme from KB cells that expresses both DNA polymerase a and a tightly coupled DNA primase activity (2, 3) and contains a number of polypeptide components. In the present studies, these antibodies form the basis of a discriminating immunoassay with which we have mapped to the short arm of the human X chromosome a single genetic locus that is necessary and sufficient for DNA polymerase a
expression.
MATERIALS AND METHODS Rodent-Human Somatic Cell Hybrids. Mouse-human hybrid clones 28-, 7-1, 10-9, 13-13, 1-2, and 45-2-3 originated from independent fusions of concanavalin A- or pokeweed mitogen-activated human lymphocytes with the hypoxanthine phosphoribosyltransferase (HPRT)-deficient mouse AKR thymoma cell line (BW 5147) (4). The CF series of hybrid clones are from fusions of the HPRT-deficient mouse cell line A9 with human fibroblasts. CF60-24 is derived from normal human fibroblasts (5). The other CF hybrid clones are derived from human fibroblasts containing X/autosome translocations: CF25-8 contains an X/13 translocation, retained der(X), Xqter-*Xp22.1 (6); CF37-6 contains an X/11 translocation (GM1695, from the Human Mutant Genetic Cell Repository, Camden, NJ) and was from a patient with Duchenne muscular dystrophy, retained der(X), Xqter -*Xp2l.2 (7); CF31-24 and CF31-4 both contain an X/20 translocation with der(X), Xqter-+Xcen (6); and CF32-23 contains an X/12 translocation with der(X), Xqter- oXq22 (8). Clone 37-26R-D-le, isolated from the CF37-6 experiment, contains a structurally normal inactive human X chromosome as the only human X-derived material (9); clone 2bR-2, isolated from the CF25-8 experiment (X/13 translocation) (6), contains der(X), Xqter-*Xp22. 1, in the inactive state in a low proportion of cells. These two clones had been selected for either spontaneous (37-26R-D-le) or 5-azacytidine-induced
RESULTS A Monoclonal Antibody Against Human DNA Polymerase a Can Distinguish Human and Rodent Polymerase Epitopes. Preliminary assessment of the anti-polymerase a monoclonal antibodies indicated that while the three neutralizing anti-
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Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; HPRT, hypoxanthine phosphoribosyltransferase. tTo whom reprint requests should be addressed.
5270
Proc. Natl. Acad. Sci. USA 82 (1985)
Biochemistry: Wang et al.
5271
100 cin LU
80
>z :3 -j00 60 0 0 a 5
w
0
40
z
Mu
20
cc 4J
0
0
I
I
I
I
25
50
75
100
SJK 132-20 IgG (ng)
0
25
50
75
SJK 237-71 IgG (ng)
100
w
I
0
20
0
40
'
60
80
100
PERCENT OF HUMAN DNA POLYMERASE a PRESENT
FIG. 1. Reactivity of monoclonal antibodies against human DNA polymerase a with human and rodent polymerase a fractions. (A) Capability of neutralizing monoclonal antibody SJK 132-20 to bind both human and mouse DNA polymerase a. One unit of DNA polymerase a (fraction 11A) (2) from KB cells or murine A9 or BW cells was incubated with neutralizing antibody SJK 132-20, and surviving polymerase a activity in the supernatant fraction was measured as described (1). e, Percent of human polymerase a bound; a, percent of murine polymerase a bound. (B) Selective binding of monoclonal antibody SJK 237-71 to human DNA polymerase a. *, Percent of human polymerase a bound; o, percent of murine polymerase a bound. (C) Reconstitution experiment to show percent of DNA polymerase a activity bound by SJK 237-71 IgG in mixtures of human and mouse polymerase a fractions. Human KB DNA polymerase a was mixed with mouse BW polymerase a in the various proportions indicated on the abscissa at a final concentration of 1 unit of total polymerase a activity per incubation. Assays were performed as in B. e, Percent of total polymerase a activity bound by 50 ng of SJK 237-71 IgG; o, by 10 ng of this IgG.
bodies extensively crossreacted with polymerase a fractions from a variety of mammalian sources, recognition by the several nonneutralizing antibodies was tightly restricted to human polymerase a epitopes. Thus, neutralizing antibody SJK 132-20 interacts with both human and murine polymerase a fractions (Fig. lA); in contrast, nonneutralizing antibody SJK 237-71 (1, 2), which has strong affinity for human polymerase a, does not interact with rodent polymerase a fractions prepared from murine thymoma cells (Fig. 1B) or from murine fibroblasts or Chinese hamster cells (data not shown). In 36 independent assays, the fraction of initial rodent polymerase a activity that was recovered in the immune pellet was 1.48% 0.97% (mean 2 SD), a value that we take as background for this assay. The specificity and sensitivity of the binding interaction of SJK 237-71 IgG with human polymerase a is further described in the reconstruction experiment shown in Fig. 1C, in which the proportion of initial polymerase a activity that was bound by the antibody (either 10 or 50 ng of IgG) was linearly correlated with the proportion of human polymerase a in the starting mixture. Control values for nonspecific binding of polymerase a, obtained from 73 independent assays performed either with no SJK 237-71 IgG or with monoclonal nonimmune P3 IgG (1), were 1.63% 0.99% (mean 2 SD) of initial polymerase a activity recovered in the immune precipitate. These control values for nonspecific and background binding provide a ±
±
±
±
rigorous exclusion limit and permit confident ascertainment of human polymerase a activity by this assay. Thus, we define values of -2.6% of total polymerase a activity recovered in the immune complex as negative for the presence of the human enzyme; in fact, all of the assays interpreted as positive gave values that were .3 times this exclusion boundary. Expression of Human DNA Polymerase oa in Mouse-Human Hybrid Clones. Seven hybrid clones, derived from five independent hybridizations and containing different complements of human chromosomes (4), were screened for the expression of human DNA polymerase a activity by the immune binding assay using SJK 237-71 IgG, and they were simultaneously karyotyped (Table 1). Five of the hybrids that contained human chromosomes 4, 5, 6, 7, 8, 10, 11, 14, 17, 18, and 21 were negative for human polymerase a activity, while clone 13-13, which contained only the human X chromosome by karyotyping, and clone CF60-24, which contained human chromosomes 4, 12, 13, 17, 19, 21, and X (5), were both strongly positive. For closer analysis, two clones, 1-2 and 28-1, which each contained a human X chromosome but not a Y chromosome in a proportion of their cells, were separated into X chromosome-positive and -negative fractions by using a fluorescence-activated cell sorter (18) and a monoclonal antibody, 12E7, specific for a cell surface determinant coded for by the human X and Y chromosomes (19, 20). The sorted
Table 1. Expression of human DNA polymerase a activity in mouse-human hybrid clones % of total DNA No. of cells (of 20 examined) containing chromosome human a bound polymerase Hybrid by SJK 237-71 clone 4 5 6 7 8 10 11 12 13 14 17 18 19 21 X 1.3 11 5 1 1 11 1 16 13 7 28F-7 1.4 7 6 9 13 5 1 6 1 28G-7 1.4 14 11 1 16 12 13 2 10 3 28D-7 1.4 15 7-1 1.4 13 10-9 8.3 6 13-13 14 3 16 16 3 4 8 CF60-24 7
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Proc. Natl. Acad. Sci. USA 82 (1985)
Table 2. Expression of human DNA polymerase a activity in hybrid clones sorted for human chromosome X No. of cells (of 20 examined) containing % of total DNA human chromosome polymerase a bound 5 2 3 4 7 12 14 Hybrid clone 6 10 11 18 21 X by SJK 237-71 1-2 11 Positive fraction 9 Negative fraction 0 0 0 0 0 0 0 0 0 0 0 0 0 1.2 28-1 5 6 1 11 9 1 4 1 8 1 10 Positive fraction 8.5 6 13 1 4 1 5 4 1 15 6 4 6 Negative fraction 9 2
cells were cultured in DMEM and simultaneously assayed for human DNA polymerase a activity and karyotyped. The results (Table 2) clearly confirm the correlation between the presence of the human X chromosome and human DNA polymerase a activity. Counterselection of the Human X Chromosome Abolishes Expression of Human Polymerase a Activity. Since the parental murine cell lines for all of the mouse-human hybrids used in these experiments are HPRT deficient, the human X chromosome can be selectively maintained or eliminated by propagating hybrid clones in HAT medium or 6TG medium, respectively (17). Three hybrid clones were subjected to medium selection and counterselection (Table 3), were karyotyped, and were extensively analyzed for the presence of human X chromosome marker enzymes, for expression of human DNA polymerase a activity, and for the presence of human DNA polymerase a antigen. When maintained in HAT medium, all three hybrids expressed the two marker enzyme activities as well as human polymerase a activity, and they contained human polymerase a antigen. In contrast, when propagated in 6TG medium, all of these functions were lost concordantly. Although hybrid clone 45-2-3 had been maintained in HAT medium and expressed human DNA polymerase a antigen and enzyme activity, as well as human G6PD and HPRT activities, it was found to contain no detectable human chromosomes by karyotyping. Counterselection of this clone in 6TG medium resulted in the survival of -5% of the starting cell population, and these cells, after serial propagation in 6TG medium, demonstrated concordant loss of human polymerase a activity and antigen, G6PD, and HPRT, suggesting that at least a fraction of the population in HAT medium contains a free and presumably highly rearranged human X chromosome that cannot be recognized by karyotyping. The three hybrid clones described in Table 3 were further evaluated for their content of human genomic DNA by a qualitative hybridization assay with a human Alu sequence probe (Fig. 2). DNA samples from the murine cell lines BW or A9 contained no hybridizable human Alu sequences. The results obtained with hybrid clones 13-13 and CF60-24, main-
tained
in
HAT and 6TG media, respectively,
are
in excellent
agreement with the karyotype data in Table 3. Note that hybrid
clone 45-2-3, while maintained in HAT medium, contained human Alu sequences at a relative abundance comparable to that in clone 13-13, whereas after counterselection in 6TG medium, human Alu sequences were no longer present. These hybridization data thus provide confirmation of the interpretation that a morphologically unrecognizable, segregatable X chromosome derivative is present in this clone. On the basis of the data presented in Tables 1, 2, and 3 and Fig. 2, we conclude that there is an absolute correlation between the expression of human DNA polymerase a activity and the presence of human X chromosomal sequences. The genetic locus for polymerase a can thus be assigned to this chromosome. Regional Mapping of the DNA Polymerase a Gene on the X Chromosome. More precise localization of the polymerase a gene on the X chromosome was accomplished with a panel of mouse-human hybrid cell lines that carry specific structural abnormalities of this chromosome, including X-autosome translocations with well-characterized break points, and one particularly informative interstitial deletion of most of band Xp2l. These experiments are summarized in Fig. 3, in which we present for each of the hybrid clones examined the structure of the human X chromosome it contains, as well as the results of assays for the expression of human DNA polymerase' G6PD, and HPRT activities. In addition, two clones were examined, 37-26R-D-le and 2bR-2, that contained only an inactivated human X or der(X) chromosome but had been selected for either spontaneous reactivation of the human HPRT locus (37-26R-D-le) or 5-azacytidineinduced reactivation of that locus (2bR-2) (10, 11). Thus, all of these hybrid clones were routinely maintained in HAT medium. The results of the standard assays for expression of human polymerase a activity with clones containing X/autosome translocations directed our attention to the short arm of the X chromosome, to the region Xp2l.2-Xp22.1, which is the shortest region of overlap between the strongly positive clone CF25-8 and the negative clone CF37-6. Final specification of a,
Table 3. Cytogenetic and biochemical analyses of hybrid clones in HAT and 6TG media No. of cells Biochemical and immunological analyses (of 20 examined) % of total DNA Presence of human containing human chromosome Human enzyme activities DNA polymerase polymerase a bound a antigen Medium 4 Hybrid clone 13 17 19 21 X by SJK 237-71 G6PD HPRT HAT 6 CF60-24 4 6 4 8 12 16.5 + + + 6TG 2 12 2.4 13-13 HAT 12 7.5 + + + 6TG 0 0 0 0 0 0 1.5 45-2-3 HAT* 0 0 0 0 0 0 22 + + + 6TG 0 0 0 0 0 0 2.4 *Hybrid clone 45-2-3 maintained in HAT medium is interpreted to contain a free, segregatable human X chromosome that is highly rearranged and unrecognizable by karyotyping (see text).
Biochemistry: Wang et al. BW
KB
lo-
1.0
4w
2.1
4w
3.8 4.1 5.5 9.0
40
Proc. Natl. Acad. Sci. USA 82 (1985) 13-13(HAT)
A9
4000
m
m
x VI,
x
+1
+
0.2
o0.3 -~ 0.4
0
OW
N
-
-
0.7
-
4_
0.1
0.3
0.6 0.8
_W 4_ 4_
_
2.1
1.2
45-2-3(6TG) CF6O24(6TG)
13-13(6TG)
0.2 0.4 0.6 1.0
0.9
1-
CF6O-24(HAT)
45-2-3(HAT)
5273
0.2
(I,
-
o
0.3 0.5
X
1.4 2.6 2.7
4 -a
0.9
+1
N
_0
1.4
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1.9
FIG. 2. Detection of human genomic DNA sequences in mouse-human hybrid clones. Single-stranded DNA, 0.3, 0.6, 1.2, 2.4, 5.4, and 8.0
jug, from human KB cells, mouse BW or A9 cells, and mouse-human hybrid clones 13-13, 45-2-3, and CF60-24, cultivated in either HAT or
6TG medium, was slot hybridized (21) with 2.2 x 10' cpm of human Alu sequence (22). The relative radioactivities of the slots, measured by Cerenkov counting, are expressed in arbitrary numerical units, with 0.3 ,ug of KB DNA defined as 1 unit of hybridization (1 unit = 15,230 cpm). Values for DNA samples from mouse BW or A9 cells and hybrid 13-13 and 45-2-3 grown in 6TG medium are presented as mean SD. ±
the genetic locus of DNA polymerase a was accomplished through study of the hybrid XVIII-54A-2a, which contains an interstitial deletion of Xp in the active state and proved to be strongly positive for human polymerase a activity. We could thus exclude most of band Xp2l and assign the gene for DNA polymerase a to the junctional region of bands Xp22.1 and Xp21.3 or, more likely given the extent of the deletion, to the band Xp22.1. Finally, to determine whether this locus might be one of those that escape X inactivation, we made use of the hybrids 37-26R-D-le and 2bR-2 (Fig. 3), which contained, respectively, an intact inactive X chromosome and the inactive der(X) homologue of the active der(X) chromosome in clone CF25-8, which is positive for the expression of human
Hybrid Clones XVNI-54A-2a CF25-8 Human pol c Human G-6-PD Human HPRT 22.3 22.2
21.3 21.2
p
21.1
11.4 11.3 11.23 11.22 11.21
11.1 I1
12.1 12.2 13
q 21.1 21 2
DISCUSSION We have exploited an assay based on a monoclonal antibody for human DNA polymerase to map a single genetic locus that is required for the expression of polymerase a activity to the region of the junction of bands Xp21.3 and Xp22.1, on the short arm of the human X chromosome, and to demonstrate that this locus is not expressed from an inactive X chromosome. The nature of the assay is such that we cannot rigorously distinguish between the two formal possibilities a
CF37-6
CF31-24
CF31 4
+
+
+
CF32-23 37-26R-D-le
2bR-2
+ +
+
1Li
A
rm
I:
polymerase a activity. Neither clone containing an inactive X chromosome expressed polymerase a activity; therefore the polymerase a locus does not escape X inactivation.
I
21.3 22.1 22.2 22.3 23 24 25 26 27
28
FIG. 3. Assignment of the genetic locus of DNA polymerase a to the short arm of chromosome X near the junction of bands Xp21.3 and Xp22.1. The idiogram of the trypsin-Giemsa banding pattern of the human X chromosome is presented at the left (23). The hatched pattern designates an inactive X or der(X) chromosome. All hybrid clones were analyzed for human DNA polymerase a expression by SJK 237-71 binding assays and for the presence of human G6PD and HPRT activities as in Table 3. A + represents 14% of total polymerase activity bound by SJK 237-71 in CF25-8 cells and 11% in XVIII-54A-2a cells, presence of human G6PD or HPRT activity of >16 nmol of IMP produced per hr per 10' cells; a represents
