Immunological Reagents for Comparisons of DNA Polymerase-cx and DNA Polymerase-/3*

(Received for publication, July 3, 1980, and in revised form, September 12, 1980)

Lucy M. S. Chang and F. J. Bollum From the Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20014

Antibodies to homogeneous calf thymus DNA polym- erase+ and calf thymus DNA polymerase-a prepara- tions were raised in rabbits. The antiserum against calf thymus DNA polymerase-j3 cross-reacts with all verte- brate DNA polymerase-/3 preparations tested, but does not cross-react with trypanosome DNA polymerase-/?, DNA polymerase-y, terminal transferase, yeast DNA polymerases, and Escherichia coli DNA polymerase 1. The antibodies against calf thymus DNA polymerase- a cross-react with DNA polymerase-a from mouse, hu- man, and chicken, but do not cross-react with DNA polymerase-a from sea urchin embryos and Drosophila embryos, DNA polymerase-/?, DNA polymerase-y, ter- minal transferase, yeast DNA polymerases, and E. coli DNA polymerase I.

Immunological reagents provide versatile and sensitive probes for detection of differences and (similarities) within protein molecules of similar function isolated from different species. Immunological comparisons are especially useful when relatedness needs to be examined with impure enzyme preparations. For example, surveys for molecular weight and reaction characteristics can be carried out to examine the gross phylogenetic relationships of the DNA polymerases (1). For closer examination of apparent abberations or evolution- ary relatedness, more specific characterization is required. Specific antibodies against DNA polymerase-/3 have been used to demonstrate that trypanosomes do indeed have DNA polymerase-/3 (2). This was an unexpected finding since free- living protozoa have been shown not to have this enzyme (1, 3, 4). Use of specific antiserum permitted the demonstration that the DNA polymerase$ was trypanosoma1 and not a contamination from the rodent host.

We have prepared several DNA polymerase-a antibodies in the past and more recently, DNA polymerase-/3 antibodies. Some of the comparisons that can be performed using these materials are described in this report. The results show the qualitative and quantitative similarities between DNA polym- erase-/3 preparations by precipitation and enzyme neutraliza- tion tests. Similar comparisons between DNA polymerase-a preparations are made. Demonstration of the differences be- tween DNA polymerase-/3 and -a, as well as nonrelatedness of more remotely similar polymerizing enzymes, are also made. These comparisons, using conventional antibody preparations, will provide a background for use as monoclonal antibodies expressing single specificities become available.

* This investigation was supported in part by Grants CA 23262 and CA 23365 awarded by the National Cancer Institute, Department of Health, Education and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

MATERIALS AND METHODS Chemicals-Deoxynucleoside triphosphates and pancreatic DNase

I-activated calf thymus DNA were prepared as previously described (5). Calf thymus DNA and bovine serum albumin were purchased from Worthington. Poly(rA) .d(pT),,’ was prepared as previously described (6). Radioactive dNTP were purchased from New England Nuclear. All other reagents were commercial reagent grade.

Enzyme Assays-DNA polymerase-P activity was assayed in re- action mixtures containing 0.05 M Ammediol-C1 buffer at pH 8.6, 0.1 M NaCl, 0.1 mM each of dATP, dCTP, dGTP, and [methyl-,”H]dTTP at 30 to 100 cpm/pmol, 8 mM MgC12, 150 pg/ml of activated calf thymus DNA, and 100 pg/ml of bovine serum albumin. DNA polym- erase-a activity was assayed in reaction mixtures containing 0.04 M potassium phosphate buffer at pH 7.0, 0.1 mM EDTA, 1 mM 2- mercaptoethanol, 0.1 mM each of dATP, dCTP, dGTP, and [methyl- “HIdTTP at 30 to 100 cpm/pmol, 8 mM MgClz, 150 pg/ml of activated calf thymus DNA, and 100 pg/ml of bovine serum albumin.

DNA polymerase-y activity was assayed in reaction mixtures con- taining 0.05 M Tris-C1 buffer a t pH 8.0, 0.15 mM [methyl-”HIdTTP a t 100 cpm/pmol, poly(rA).d(pT),, (0.1 mM adenylate complexed with 0.01 mM thymidylate), 0.5 mM MnCl,, I mM dithiothreitol, 0.1 M KCl, and 100 pg/ml of bovine serum albumin.

Terminal deoxynucleotidyltransferase (7) and yeast DNA polym- erases (8) were assayed as previously described. Escherichia coli DNA polymerase I was assayed using DNA polymerase-a conditions. All incubations were carried out a t 35°C and products of the reaction were detected as acid-insoluble radioactivity (5). One unit of enzyme is defined as 1 nmol of total nucleotide polymerized/h.

DNA Polymerase-&-For preparation of the antiserum to DNA polymerase-P, homogeneous calf thymus DNA polymerase-P was used. From 8 kg of calf thymus glands, 750 pg of pure protein were obtained (5). The DNA polymerase-P preparations from human liver, calf liver, and chick embryo were prepared by the same procedure used for the calf thymus enzyme. Mouse myeloma DNA polymerase- /3 was a generous gift from Dr. S. Wilson, National Institutes of Health, Bethesda, MD. DNA polymerase-P from other sources was not extensively purified, although all of the DNA polymerase-,8 sources used were free of other deoxynucleotide-polymerizing activi- ties.

DNA Polymerase-a-Calf thymus DNA polymerase-a was pre- pared from a partially purified enzyme fraction, previously described as the DNA polymerase-a pool from a DE-11 cellulose column (9). The DNA polymerase-a pool from the DE-11 cellulose column was stored at -20°C after precipitation from solution with (NH4)zSOd followed by resuspension in 0.05 M potassium phosphate at pH 7.4. For a typical fractionation, 8 ml of starting fraction were dialyzed overnight against 0.05 M Tris-Cl buffer at pH 7.8 containing 1 mM 2- mercaptoethanol and 0.5 M NaCl. The dialyzed enzyme was fraction- ated on a Sephadex G-200 column (2.6 X 90 cm) equilibrated with the dialysis buffer. The active fractions (53 ml) from the Sephadex G-200 column were pooled and potassium phosphate at pH 7.2 ( 1 M) was added to bring the solution to 0.05 M potassium phosphate. The Sephadex G-200 fraction was then loaded onto a hydroxylapatite (10) column (1.5 x 7 cm) equilibrated with 0.5 M NaCI, 1 m M 2-mercap- toethanol in 0.05 M potassium phosphate at pH 7.2. After loading, the column was washed with 25 ml of the equilibration buffer and then eluted with a linear potassium phosphate gradient (150 ml) from 0.05 to 0.2 M potassium phosphate (pH 7.2) containing 0.5 M NaCl and 1

I The abbreviations used for nucleotides and polynucleotides are those of the IUPAC-IUB Commission and Biochemical Nomenclature (CBN) (1970) J. Biol. Chem. 245, 5171-5176.

494

Antibodies to Mammalian DNA Polymerases 495

mM 2-mercaptoethanol. DNA polymerase-a activity eluted from this hydroxylapatite column as a sharp peak at 0.135 M. The active fractions from the hydroxylapatite column (18 ml) were pooled and precipitated by dialysis against 70% saturated in (NH,),SO< in 0.05 M potassium phosphate a t pH 7.2. The (NH,),S04 precipitate was collected by centrifugation, redissolved in 0.5 M NaCI, 1 mM 2-mer- captoethanol, 0.05 M Tris-CI at pH 7.8 in 50% glycerol, and stored at -20°C.

DNA polymerase-a from HeLa cells, mouse L-cells, and chick embryos were not as extensively purified. Soluble extracts (0.2 M potassium phosphate a t pH 7.4) of these sources were purified by chromatography on phosphocellulose followed by chromatography on DE-I1 cellulose and hydroxylapatite. The sea urchin embryo DNA polymerase-@ and Drosophila embryo DNA polymerase-a used were prepared by fractionation of the soluble extract on a 5 to 20% sucrose gradient in the presence of 0.5 M NaCI.

Other Deoxynucleotide-polymerizing Enzymes-Calf liver DNA polymerase-y was a generous gift from Dr. S. Wilson, National Insti- tutes of Health, Bethesda, MD. Calf thymus terminal deoxynucleo- tidyltransferase (11) and yeast DNA polymerases (8) were purified as previously described. E. coli DNA polymerase I was a generous gift from Dr. L. Loeb, University of Washington, Seattle, WA.

Preparation of Antiserum to Calf Thymus DNA Polymerase-@- Homogeneous calf thymus DNA polymerase-@ was modified by cross- linking with glutaraldehyde and the modified enzyme was used to immunize the animal. Cross-linking was carried out by treating 100 pg/ml of calf thymus DNA polymerase-@ in phosphate-buffered saline with 0.1% glutaraldehyde for 20 min at room temperature. Excess glutaraldehyde was destroyed by addition of NaBH, to 0.0570.

A male New Zealand rabbit (4 kg) was used for immunization. Primary immunization was made at the footpads with 50 pg of cross- linked enzyme (0.5 ml) suspended in 0.5 ml of Freund‘s complete adjuvant. A secondary immunization consisting of 50 fig of the cross- linked enzyme protein in Freund’s incomplete adjuvant was admin- istered a t multiple subcutaneous sites 2 weeks after the primary injection. Four successive boosters (50 pg each of the cross-linked enzyme) were administered at 2-week intervals. Two weeks after the fourth booster, a booster of 100 pg of unmodified calf thymus DNA polymerase-@ in Freund’s incomplete adjuvant was administered and this was followed by two additional boosters of 65 pg of the cross- linked enzyme a t 2-week intervals. Test bleedings were done 11 days after each injection. The final antiserum used for this study was obtained after the fourth booster with the cross-linked antigen. Titer is defined as the dilution of 10 p1 of the antiserum required to inhibit 50% of 1 unit of DNA polymerase activity.

Preparation of the Antibodies to Calf Thymus DNA Polymerase- a-Both purified calf thymus DNA polymerase-a (Ag-I) and an immune complex (Ag-11) of Ag-I and an antibody preparation to calf thymus DNA polymerase-a (Ab-I) were used to generate the anti- bodies to calf thymus DNA polymerase-a for this study (Ab-11). The Ag-I1 was prepared by precipitation of 1.5 mg of Ag-I with 50 mg of Ab-I previously prepared in our laboratories (12). Ab-I was produced in a rabbit using a partially purified calf thymus DNA polymerase-a preparation purified essentially as described by Yoneda and Bollum (13), except that an additional gel filtration column on Sephadex G- 100 was included to remove low molecular weight polymerizing activ- ities. The precipitation reaction between Ag-I and Ab-I was carried out in phosphate-buffered saline for 1 h at 35°C followed by incuba- tion at 4’C for 16 h. The immune precipitate was collected by centrifugation and was washed six times by suspension in phosphate- buffered saline followed by centrifugation. The washed immune com- plex (Ag-11) was then suspended in 0.5 ml of phosphate-buffered saline and 0.5 ml of Freund’s incomplete adjuvant, and used in the immunization schedule.

Antiserum to calf thymus DNA polymerase-a described in this report (Ab-11) was obtained from a male New Zealand rabbit (4 kg). After the rabbit was bled to obtain the control serum, primary immunization was made at multiple subcutaneous sites with 1 mg of Ag-I (0.5 ml) suspended in 0.5 ml of Freund’s complete adjuvant. A secondary immunization consisting of 1 mg of Ag-I in Freund’s incom- plete adjuvant was administered 3 weeks after the primary injection. Three weeks after the secondary immunization, Ag-11 in Freund’s incomplete adjuvant was injected subcutaneously and the rabbit was bled at 11 days and 19 days after this booster. The control and antisera obtained were diluted with an equal volume of phosphate- buffered saline and the crude immunoglobin was obtained by precip- itation of the sera twice a t 50% saturation (NH4),S04.

Enzyme Neutralization Reactions-DNA polymerases were di-

luted to 50 to 200 units/ml with 1 mg/ml of bovine serum albumin in phosphate-buffered saline. The diluted enzyme (10 pl) was incubated with 10 pl of antiserum dilution (in 1 mg/ml of bovine serum albumin in phosphate-buffered saline) for 30 min at room temperature and overnight at 4OC. The enzyme activity remaining after incubation was assayed as described above and compared to the equivalent dilution of control serum or immunoglobin. The control lines are horizontal with the antibodies used. Undiluted serum sometimes gives a slight (5 to 10%) stimulation of DNA polymerase activity. Properly collected serum from control animals or from animals immunized with a variety of antigens does not interfere with the DNA polymerase reactions as carried out.

Immunodiffusion and Immunoelectrophoresis-Immunodiffusion was carried out on microscope slides coated with 1% agarose in phosphate-buffered saline. Antigens to be tested contained at least 5000 units/ml of DNA polymerase activity. Diffusion was carried out in a moist chamber for 6 h a t room temperature, a t which time direct visualization of precipitation lines was possible. The slides were then washed for 24 h with several changes of phosphate-buffered saline, 2 h with three changes of distilled water, and then air-dried. After drying, the precipitin lines were visualized by staining with Coomassie blue in methanol and acetic acid.

Immunoelectrophoresis was carried out using an LKB immuno- electrophoresis apparatus. Electrophoresis was run in 1% agarose gels containing 0.06 M Tris/barbital/sodium barbital buffer (Gelman High Resolution buffer) at pH 8.8 for 3 h at 200 V. After electrophoresis, immunoglobin G solutions were added to the antisera troughs and diffusion was allowed to take place in a moist chamber overnight a t room temperature. The resultant precipitin lines were photographed directly with a Cordis immunodiffusion camera.

RESULTS

Antiserum to Calf Thymus DNA Polymerase-/?

Response of the Rabbit to the Immunization-After three successive injections with the cross-linked calf thymus DNA polymerase-/?, the animal developed a weak antiserum with a titer of 1/6 against calf thymus DNA polymerase-/?. Three additional boosters with the cross-linked antigen raised the titer of the antiserum to 1/28. Additional boosters with un- modified antigen or cross-linked antigen produced no increase in titer. The antiserum obtained after six injections with the cross-linked antigen was used for comparative studies.

Effects of Calf Thymus DNA Polymerase-/? Antiserum on Other Eukaryotic Deoxynucletide-polymerizing Enzymes- The rabbit antiserum produced against homogeneous calf thymus DNA polymerase-/? is completely specific for DNA polymerase-/? at the sensitivity of testing we have used. No effect on the activities of mammalian DNA polymerase-a, -y, and terminal transferase, nor yeast DNA polymerases and E. coli polymerase I (Table I) is observed. The neutralization titers of the DNA polymerase-/? antiserum vary with the source of DNA polymerase-/? with the cross-reactivity de- creasing with decreasing relatedness of the organisms (Table I). The titer of the antiserum against calf thymus DNA polymerase-/? is three times higher than that against chick embryo DNA polymerase-/?, and is 14 times higher than toadfish DNA polymerase-/? (Table I and Fig. 1). No enzyme inhibition was observed with calf thymus DNA polymerase- /? antiserum on DNA polymerase-/? from T. brucei, a parasitic protozoan (2).

The antibody prepared against calf thymus DNA polym- erase-/? is a precipitating antibody. A single precipitin line is observed with the antiserum and its homologous antigen (Fig. 2, a and e). The immunodiffusion technique can also be used to study the relatedness of DNA polymerase-/? from other biological sources. A line of identity can be observed with human liver DNA polymerase-P (Fig. 2b). A weak precipitin line is detected with chick embryo DNA polymerase-/? (Fig. 2d); this weak line, nevertheless, is also a line of identity. No precipitin line was found with DNA polymerase-/? from toad-

496 Antibodies to Mammalian DNA Polymerases

TABLE I Effects of calf thymus DNA polymerase-P antiserum on various

deoxynucleotide-polymerizing enzymes Enzyme Titer“

Calf thymus DNA polymerase-l( 1/28 Calf liver DNA polymerase-l( 1/26 Human liver DNA polymerase-P 1/20 Rabbit liver DNA polymerase-/3 1/18 Rat liver DNA polymerase-P 1/18 Dog liver DNA polymerase-P 1/17 Mouse myeloma DNA polymerase-l( 1/12 Chick embryo DNA polymerase-/3 1 /9 Toadfish DNA polymerase+ 1/2 Trypanosoma brucei DNA polymerase-P No inhibition Calf thymus DNA polymerase-a” No inhibition Calf thymus terminal transferase No inhibition Dog liver DNA polymerase-y No inhibition Calf liver DNA polymerase-y No inhibition Yeast DNA polymerase I No inhibition Yeast DNA polymerase I1 No inhibition E. coli DNA polymerase I No inhibition The titer is defined as the dilution of antiserum (IO p l ) required

to inhibit 50% of 1 unit of DNA polymerase activity. The dilutions shown were calculated from the titration curve.

‘Other preparations of DNA polymerases-a from HeLa cells, mouse L-cells, and chick embryo were also negative.

1 Dilution of Antiserum

FIG. 1. Calf thymus DNA polymerase-/I antiserum neutrali- zation of DNA polymerase-/I from various sources. Enzyme neutralization assays were carried out as described under “Materials and Methods.” The amounts of enzyme used were 1.29 units for calf thymus DNA polymerase-/3 (A-A), 1.27 units for chick embryo DNA polymerase+ (m), and 0.91 unit for toadfish DNA polym- erase-p (A-A).

fish and T. brucei (data not shown). No precipitating cross- reactivity was demonstrated with the antiserum to calf thy- mus DNA polymerase-P and calf thjrlnus DNA polymerase- a (Fig. 2e) or calf thymus terminal transferase (Fig. 2f ).

Antiserum to Calf Thymus DNA Polymerase-a Preparation of Calf Thymus DNA Polymerase-a for Im-

munization-Since homogeneous DNA polymerase-a from calf thymus glands is not available, immunological subtleties were used to prepare the antiserum to DNA polymerase-a. The antibody to DNA polymerase-a produced for this study was a “second generation antibody,” that is, an antibody elicited against the antigen in an immune complex. The “fmt generation antibody” (Ab-I) was prepared in rabbits using calf thymus DNA polymerase-a (Ag-0) prepared essentially as described by Yoneda and Bollum (13). Ab-I produced multiple precipitin lines against Ag-0. Ag-I was a more highly purified calf thymus DNA polymerase-a and was prepared by a mod- ified purification procedure. This procedure is outlined under “Materials and Methods.” A summary of the purification scheme for Ag-I is outlined in Table 11. Purified Ag-I has a specific activity of -22,000 and it is -1,500-fold purified from

f

a b

S C

e d

FIG. 2. Immunodiffusion with calf thymus DNA polymerase- /I antiserum. Immunodiffusion was carried out as described under “Materials and Methods.” The center well (s) contained the antise- rum. Other wells are: a, calf thymus DNA polymerase-P; b. human liver DNA polymerase-l(; c, calf thymus DNA polymerase-l(; d, chick embryo DNA polymerase-P; e, calf thymus DNA polymerase-a; and f, calf thymus terminal transferase.

TABLE I1 Purification of DNA polymerase-a from calf thymus glands

Specific ac- Total en- Fraction tivity zyme activity

mR units/rng DE-11 cellulose pool 120 540 w800 Sephadex G-200 pool 23 2,670 61,800 Hydroxylapatite pool 1.9 21,200 40,700

.a

f

b

?

\ S C

e d

l

FIG. 3. Immunodiffusion with antibodies to calf thymus DNA polymerase-a. Immunodiffusion was carried out as described under “Materials and Methods.” The center well (s) contained Ag-I, the calf thymus DNA polymerase-a preparation as described under “Materials and Methods.” Other wells are: a, Ab-I; b, Ab-11; c, phosphate-buffered saline; d, calf thymus DNA polymerase-P; e, Ab- 11; and f, calf thymus terminal transferase. See “Materials and Meth- ods” and text for information on antigen and antibody preparations used.

the soluble calf thymus extract. Only one precipitin line can be detected by immunodiffusion (Fig. 3a) and immunoelectro- phoresis (data not shown) of the Ab-I against Ag-I. The washed immune complex of Ag-I and Ab-I (Ag-11) should contain only DNA polymerase-a since all DNA polymerase- a activity was removed during the precipitation reaction.

Response of the Rabbit to the Immunization-After two injections with Ag-I, no immunoprecipitin line was detected with the test serum and Ag-I. Eleven days after the booster with the Ag-11, a sharp immunodiffusion line was detected with the test serum and Ag-I. On the 19th day after the Ag-I1 booster, the rabbit was exsanguinated and its serum was

Antibodies to M a m m a l i a n DNA Polymerases 497

TABLE I11 Effects of calf thymus DNA polymerase-cy antiserum on various

deoxynucleotide-polymerizing enzymes Enzyme Titer”

Calf thymus DNA polymerase-a 0.13 Human HeLa cell DNA polymerase-cy 1.6 Mouse L-cell DNA polymerase-a 2.2 Chick embryo DNA polymerase-a 2.4 Sea urchin embryo DNA polymerase-cy No inhibition Drosophila embryo DNA polymerase-a No inhibition Calf thymus DNA polymerase-gh No inhibition Calf thymus terminal transferase No inhibition Calf liver DNA polymerase-y No inhibition Yeast DNA polymerase I No inhibition Yeast DNA polymerase I1 No inhibition E. coli DNA polymerase I No inhibition The titer is defined as micrograms of immunoglobin G required

to inhibit 50% of 1 unit of DNA polymerase activity. The amounts of immunoglobin G shown were calculated from the titration curves.

All other DNA polymerase-P preparations listed in Table I were also tested and were not inhibited.

processed for isolation of immunoglobulin (Ab-11) as described under “Materials and Methods.”

Properties of Antibody to Calf Thymus DNA Polymerase- a-Ab-I and Ab-I1 each produces a single line of identity Ag- I by both immunodiffusion (Fig. 3, a and b) and immunoelec- trophoresis (data not shown). The titer of Ab-I1 is about four times higher than Ab-I against Ag-I by enzyme neutralization assays. Ab-I1 is a specific antibody for DNA polymerase-a since no precipitin line can be detected for calf thymus DNA polymerase-/? (Fig. 3d), nor the calf thymus terminal transfer- ase (Fig. 3f ). In neutralization assays, Ab-I1 has no inhibitory activity against DNA polymerase-/?, DNA polymerase-y, ter- minal transferase, yeast DNA polymerases, and E. coli polym- erase I (Table 111). Earlier work has shown that Ab-I does inhibit mammalian DNA polymerase-/3 (12), suggesting that Ab-I has much broader specificity compared to Ab-11.

Effects of Antibodies to Calf Thymus DNA Polymerase-a on DNA Polymerase-a from Other Eukaryotic Sources-Like the antiserum to DNA polymerase-/3, the antibodies to DNA polymerase-a also provide some information concerning the relatedness of DNA polymerase-a from a variety of eukaryotic systems. Ab-I1 to calf thymus DNA polymerase-a, like Ab-I, cross-reacts with DNA polymerase-a from human, mouse, and chicken (Table HI), although the titers of Ab-I1 on the human, mouse, and chicken enzymes are -10-fold lower than its titer on the homologous enzyme. DNA polymerase-a from more diverse organisms such as sea urchin embryos and Drosophila embryos, however, are not inhibited by the antibodies to calf thymus DNA polymerase-a. The relatedness of the calf, hu- man, mouse, and chicken enzymes can also be demonstrated with the immunodiffusion technique, since weak precipitin lines can be detected using Ab-I1 with the human, mouse, and chicken enzymes (data not shown).

DISCUSSION

This work describes the fist successful preparation of an- tibody to homogeneous DNA polymerase-P. From the im- munization schedule used, it is clear that mammalian DNA polymerase-P is not a very antigenic protein. Even with the protein modification steps previously found useful in prepa- ration of terminal deoxynucleotidyltransferase antibody (14) and multiple injections, the antiserum obtained was of modest titer. The rarity of high quality mammalian DNA polymerase- P preparations does not readily permit experiments on im-

proved immunization schedules. Since homogeneous DNA polymerase-a is not available for immunization, immunologi- cal manipulations were made to generate an antiserum to this enzyme. By all immunological criteria, the antibodies to DNA polymerase-a produced in this study are monospecific to DNA polymerase-a.

Investigations on the antibodies we have obtained demon- strated that rabbit antibody to calf thymus DNA polymerase- a shows a gradation of cross-reactivity with nonhomologous antigens. Using direct neutralization assays reactivity with avian DNA polymerase-a can be demonstrated, but no neu- tralization of Drosophila enzyme was found. Rabbit antibody to calf thymus DNA polymerase-,B also shows a graded inhi- bition of activity of homologous antigens. Direct neutraliza- tion shows cross-reactivity with Teleost DNA polymerase-/3 and a precipitin reaction is observed with avian enzyme. We believe these results are in harmony with the evolutionary relationships to be expected between similar proteins with similar activity, although other investigations have not always produced similar results.

A rabbit antibody produced against HeLa DNA polymer- ase-a showed cross-reactivity with hamster enzyme (15), whereas rabbit antibody against chick embryo DNA polym- erase-a did not neutralize the hamster enzyme (16). Rabbit anti-chick embryo DNA polymerase-/3 did show cross-neu- tralization with hamster (16) and rat enzyme (17). We believe that all studies that have been carried out demonstrate the relatedness between DNA polymerase+ species across phy- logenetic lines and that the DNA polymerase-a studies can be interpreted as demonstrating similar relatedness. In serolog- ical studies of this kind, a negative result should be interpreted more as the result of the response of an individual rabbit, rather than denial of a rather well-established generality.

The anti-sera tested in the present set of experiments show no cross-reactivity between different eukaryotic DNA polym- erase species, that is, a, ,f3, or y, within the same animal species. Previous experiments from this (12) and other (17) laborato- ries have suggested that such relationships exist. From an evolutionary point of view, one would expect this relation, at least if peptide sequences around the catalytic center are conserved. Antibody specifically directed against the catalytic center of a DNA polymerase might allow direct examination of the evolution of catalytic mechanisms in the fundamental process of DNA replication.

Antibodies to eukaryotic DNA polymerases provide useful reagents for analysis of the biological relations and biochem- ical properties of the DNA polymerases in complex cells. In the future, they may provide access to accessory proteins and messenger RNAs involved in the replication process. The wealth of technology available through such reagents justifies continued efforts in this direction.

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