J. Md. Biol. (1968) 33, 3547

p-Galactosidase : Immunological Studies of Nonsense, Missense and Deletion Mutants

AUDREE V. FOWLER AND IRTTNG ZABIN

Department of Biok@ul Chemistry U.C.L.A. School of Mdicine and Molecular Biology Institute

University of Califmnia, Los Angeles, California 90024, U.S.A.

(Received 26 June 1967, and in revised form 29 November 1967)

A series of nonsense mutants which map throughout the /?-galactosidase structural gene, and several missense and deletion mutants, were analysed for antigenic activity with purified antiserum produced from native j%galactosidase. Quantita- tive ana,lyses by the Ouchterlony test, by inhibition studies, and by micro- complement fixation are in general agreement. These show, relative to the position of the nonsense mutation, no immunological activity from mutants of the early (operator) part of the gene, a peak at the center, next a much lower level, and finally a relatively high level in mutants mapping in the terminal part of the gene. No antigenic activity was found in a deletion mutant which produces both the alpha and omega fragments of ,9-galactosidase.

Examination of precipitin lines on Ouchterlony plates of nonsense, missense and deletion mutants, as well as other data, suggests the presence of three classes of antigenic sites, those of relatively long chains, those of relatively short poly- peptide chains and, tentatively, those due to the polymer. Heat treatment or addition of sodium dodecyl sulfate destroys immunological activity of nonsense mutant protein.

It is suggested that variations in level of antigenic activity of incomplete fi-galactosidase chains are due not only to the presence or absence of antigenic sites, but in addition to differences in conformation of t,hese chains.

1. Introduction

The observation that induced cultures of many P-galactosidase nonsense mutant strains form material which cross-reacts against antiserum prepared with native p-galactosidase can be exceedingly useful in the investigation of the structure and mode of synthesis of this protein. We have already demonstrated (Fowler & Zabin, 1966) that the approximate size of incomplete polypeptide chains produced by four different nonsense mutants is in excellent agreement with the size predicted from genetic mapping (Newton, Beckwith, Zipser & Brenner, 1965) of these same strains.

Furthermore, we have found that extracts of several strains in which the position of the mutation is near the operator-distal (permease) end of the 2 @galactosidase) gene contain relatively high levels of antigenic activity, whereas an extract of a mutant which maps near the operator-proximal end of the Z gene exhibited no immunological activity to anti-p-galactosidase. These results provide evidence for orientation of the protein to its gene in such a manner that the coding sites for ammo- and carboxyl- terminal ends of /3-galactosidase correspond to operator and permease ends of the 2 gene, respectively.

35

36 A. V. FOWLER AND I. ZABIN

The present report gives a detailed investigation of some of the immunological characteristics of incomplete /I-galactosidase polypeptide chains. For comparison, certain deletion mutants and missense mutants have also been examined. The pro- duction of cross-reacting material by a spectrum of nonsense mutants which map throughout the 25 gene has been measured by the Ouchterlony test, by inhibition studies and by micro-complement fixation, and some experiments have been carried out showing dependence of immunological activity on “native” three-dimensional structure of incomplete chains.

2. Materials and Methods o-Nitrophenyl-fi-n-galactoside was purchased from Calbiochem, and isopropyl-/?-n-

thiogalactoside from Cycle Chemical. Guinea pig complement was obtained from Hyland Laboratories, and sheep red blood cells and hemolysin were kindly supplied by Dr Eli Sercarz. /&Galactosidase was isolated as described earlier (Karlsson et al., 1964).

(a) Bacteriul growth conditions and preparation of extracts

Bacterial strains were generously supplied by Drs Jonathan R. Beckwith, Francois Jacob, David Zipser and Michael Malamy. The relative positions of the nonsense mutations and the deletion mutations (Newton, Beckwith, Zipser & Brenner, 1965; Ulhnann, Jacob L Monod, 1967) are reproduced in Fig. 1. For convenience, we have divided the map into 16 areas of equal length, but it should be noted that these are only approximate (Newton et al., 1965). Properties of the two missense mutants, Z, and ZuIT8, have been described by Perrin (1963). Cultures were grown at 30°C with aeration in mineral medium 63 (Herzen- berg, 1959) containing 0.4% glycerol and amino acids at 50 rg/ml., thiamine at 1 rg/ml. and 5 x 10Y4 M- or low3 M-isopropyl-fl-n-thiogalactoside, as required. Bacteria were harvested in the exponential phase of growth, washed with 0.05 M-potassium phosphate buffer (pH 7.2), and were broken by brief treatment in a sonic oscillator. Clear extracts were obtained by centrifugation at 48,000 g for 30 min.

(b) Preparation of antisera to fl-galmtmid~e

Complete Freund’s adjuvant containing 2 mg of b-galactosidase was injected into the foot pads of a rabbit. After 4 weeks, the rabbit was bled and was given 10 mg of /3-galacto- sidase in 0.05 M-potassium phosphate buffer (pH 7.2) intravenously. Bleedings of about 25 ml. were taken 7, 9 and 13 days after the second injection; and after clotting, erythro- @es and fibrin were removed by centrifugation. We are indebted to Dr Eli Sercarz for preparing the antisera.

(c) PuriJicatim and concentration of antisera

To remove non-specific antibodies from the antisera, small portions of the latter were incubated with increasing levels of extracts of uninduced mutant X 90. After 1 hr at 37°C the mixtures were maintained at 4°C for 2 days and the precipitates were removed by centrifugation. The clear supernatant solutions were tested by double diffusion against extracts obtained from induced and uninduced cultures of wild-type or nonsense mutants. By this procedure, the level of uninduced X 90 extract required to remove all reaction with extracts of uninduced cultures was determined, and the calculated amount of the same extract was added to the major portion of antiserum, which was then treated in the same manner. In a typical case, 285 mg of protein were required to remove all non-speciiic antibodies from 17 ml. of antiserum.

The antibody in the clear supernatant solution was further purified by addition of 17 g of sodium sulfate for each 100 ml. The mixture was incubated at 28°C for 45 min and was centrifuged at room temperature. The precipitate, containing all the anti-/3-galactosidase, was dissolved in a small volume of 0.05 M-potassium phosphate buffer (pH 7.2) and was dialyzed thoroughly against the same buffer to remove salt. The dialyzed solution was made to a volume one-fourth that of the original antiserum, and is referred to in this paper as concentrated antiserum.

IMMUNOLOGY OF fl-GALACTOSIDASE MUTANTS 37

(d) Inzmunological procedures (i) Ouchterlony

Double diffusion in agar (Ouchterlony, 1949) W&E carried out on microscope slides and the amount of cross-reacting material was determined by dilution as previously described (Fowler & Zabin, 1966). One unit of CRMt is defined as the minimum amount in 0.01 ml. able to produce a visible precipitin line. Except as noted, the concentrated antiserum was diluted tenfold with O-05 M-phosphate buffer for these tests.

(ii) Inhibition

Quantitative inhibition studies were performed as described by Perrin (1963) with concentrated antiserum diluted loo-fold in the phosphate buffer. Tubes containing 0.1 ml. of the diluted antiserum, 1000 to 8000 units of j3-galactosidase (pure enzyme or extracts of induced Hfr 3000), extracts of mutant strains as indicated, 10 mg of bovine serum albumin and 0.05 M-potassium phosphate buffer (pH 7.2) to a final volume of 1 ml. were incubated for 1 hr at 35°C. The mixtures were maintained at 4°C for 2 days, and the /3-galactosidase activity remaining in the supernatant solution was determined after centrifugation.

(iii) Micro-cornplewzent &cation Micro-complement fixation was carried out by the method of Wasserman & Levine

(1961), except that five 50% C’F units of guinea pig complement were used instead of 1.2 units. The concentrated antiserum W&B diluted 1 :lO,OOO or 1: 35,000 in the final reaction mixtures. All components were diluted with 0.01 ivr-Tris buffer (pH 7.6) containing 0.14 M-

NaCl, 1.5x 10m4 M-Cd&, 5x 10M4 M-MgSC, and Oel’$& bovine serum albumin. The reaction mixture was maintained at 4°C for 18 hr before assaying for complement with sensitized red blood cells.

(e) Enzyme and protein assays

j3-Galactosidase was assayed by hydrolysis of o-nitrophenyl-fl-n-galactoside in PM2 buffer containing 0.05 M-mercaptoethanol (Horiuchi, Tomizawa & Novick, 1962). One unit of enzyme is defined as that amount which forms 1 mrmole of o-nitrophenol in 1 min at 28°C. Protein was measured by the Folin method of Lowry, Rosebrough, Farr & Randall (1951). Thiogalactoside transacetylase was assayed as described previously (Zabin, 1963).

3. Results

(a) Puri@ation of antiserum

With antiserum to /I-galactosidase, prepared earlier (Berg & Zabin, 1964) and stored for several years in the frozen state, extracts of uninduced cultures of several nonsense mutants gave no detectable precipitin bands by the Ouchterlony test (Fowler & Zabin, 1966). For the present experiments, fresh antiserum of higher titer was prepared, which in contrast to the earlier preparation contained antibodies to components present in extracts of uninduced cultures. However, it was possible to purify the antiserum by absorption with protein from an uninduced culture of a nonsense mutant, as described above. This treatment was successful in removing all traces of non-specific antibodies (Plate I) even when uninduced extracts were tested by double diffusion in agar with the concentrated rather than the tenfold diluted antiserum. (This procedure was not effective in purifying a sample of goat antiserum to /3-galactosidase kindly supplied by Dr M. Cohn.)

(b) Ouchterlony tests of mutant /3-galactosidase

(i) Quantitatiae assays

Table 1 shows the results of quantitative Ouchterlony assays of CRM and of thiogalactoside transacetylase produced upon induction of 20 nonsense mutants

t Abbreviation used: CRM, cross-reacting material.

38 A. V. FOWLER AND I. ZABJN

0 z ‘i

2 YA486 lJi39 % 536 Ul31 YA482 NC125 YA559 NC750 YA596 YA515 x90

Ull8 X64 NC422 X82 YA404 NC200 U336 YA623

I I I I I I I I I I I I I I I I I I I I I I I

1 2 3 4 5 6 7 0 9 IO II I I

I2 13 14 15 16 ’ i i

I 4680

4

U 163

FIG. 1. Map positions of b-galactosidase mutants.

which map throughout the 2 gene. Extracts of uninduced cultures were also ex- amined, and except for mutant strains U 366 and U 239 which are constitutive (i-), no CRM was detectable. Relative units of CRM in relation to the position of the non- sense mutation are plotted in Figure 2. The polarity effect on the production of thio- galactoside transacetylase noted by Newton et al. (1965) is clearly evident. However, the level of CRM activity does not follow the same pattern. Although mutants in

TABLE 1

Quantitative Ouchterbny analyses of cross-reacting material

Mutant TYPe Area CRM Thiogalactoside

units/mg transacetylase protein (units/mg protein)

x 90 YA 623 YA 615 U 366 YA 596 NG 200 NG 750 Y-A 404 Y-A 559 YA 536 x 82 NG126 NG 422 u 239 YA 482 X 64 YA 486 u 131

s 118 Hfr 3000 Z1 Z” 178 4680 U 163 2A 238

Ochre Ochre Amber Amber Ochre Amber Amber Ochre Amber Amber Amber Amber Amber Amber Amber Amber Amber Amber Ochre Ochre Wild Missense Missense Deletion Deletion Deletion

16 15 14 13 12 11 10

9 8 7 7 6 5 5 4 3 3 2 1 1

7 11

4-10 5-13

15 and 16

47-54 46-54 41-55 40-53-f 3141 77-93 1619

4 l&17

25 17 62

2325t 0 0 0 0 0 0

240 225

4&68 0

16 57

2.1 1.6 0.9 0.7 0.6 0.5 0.2 0.1 0.03 0.07 0.1 0.06 0.08 0.05 0.05 0.04 0.07 0.05 0.03 0.03 2-3 3.0 1.4 0.8 0.6 0.01

t The same levels of CRM and thiogalactoside transacetylase were found in uninduced cultures.

PLATE 1. Purification of antiserum. Center well (b), 10 pl. of untreated antiserum; center wt4 (a), 10 ~1. of concentrated antiserum diluted fourfold. Clockwise, starting at top around both (b) and (a), 10 ~1. of extracts of induced S 90. intlurcti I’ 118, uninducrd 1’ 118, uninduwtl S 90, induced S 90, and induced Hfr 3000.

tifr 3000 NG 125

zi 0 Zl YA 559 0 x90

0 0

0 0 Z

Ui78

0 NG 200 u239

0 2A 238

x so U 163

PLATE II. Photograph and drawing illustrating antigcnic relationships. The ccnt,rr well van- tained 10 pl. of concentrated antiserum diluted tenfold. and the outer’ wells contained 10 pl. of extracts of the indicated skains.

PLATE III. Photograph and drawing illustrating antigmic relationships. The center well COW tained 10 pl. of concentrated antiserum diluted fourfold, and the outer wells contained 10 JAI. of extracts of the indirated strains.

IMMUNOLOGY OF fl-GALACTOSIDASE MUTANTS

I 01 12’2

D C

liihiEk .

- 0.8 :v,

-0-6 g -2 o 3

-0-4 -0.2 f

-I

0 12345 6 7 8 9 IO II I2 I3 14 15 16

Map position of nonsense mutation (area)

FIQ. 2. CRM activity (bars) and thiogalactoside tranaacetylase (filled circles) in extracts of nonsense mutants. A, B, C and D are straina U 239, NG 422, X 82 and YA 538, respectively.

39

areas 11-16 have high levels and mutants of areas 14 have no CRMt in parallel with the polarity effect, there is a pronounced peak in production of CRM units in mutants around area 6, and a minimum in the mutant in area 9. This has been observed in many sets of analyses of different cultures. No differences in the behavior of amber versus ochre mutations were apparent.

Table 1 includes the results of determinations of activities for three deletion mutants, two missense mutants and a wild-type strain for comparison. Strain 4680 carries a deletion in the central section of the gene from areas PI0 and produced no CRM. However, strain U 163, in which the deletion begins in area 5 and extends into area 12, was found to yield an activity equivalent to that of the nonsense mutants which map in area 5. Strain 2A 238, in which the deletion begins in area 15 and ex- tends further into the luc operon, has a level of CRM activity equivalent to that of the nonsense mutant which maps in area 15. These data suggest that the presence of at least part of the central portion of the gene is critical for the establishment of CRM activity.

The missense mutant Z, forms a CRM which appears to exist in dimeric form, whereas that of Z, 1,,,, also a missense mutant, is a monomer (Perrin, 1963). It is of interest to note that the level of CRM activity present in extracts of Z, was found to be equivalent, to that measured by this method in the wild-type strain. On the other hand, the level of CRM activity present in extracts of Z, 1,8 was approximately equivalent to that of nonsense mutants mapping at the right end of the gene.

(ii) Antigenic similuritks

Studies presented in the preceding section were concerned with measurements of the levels of CRM activity. We have also examined carefully the nature of the pre- cipitin lines obtained in agar gels to obtain information regarding antigenic relation- ships between /3-galactosidase and the immunologically active mutant proteins. Plates II and III illustrate some of these relationships. Because details are difficult to reproduce in a photograph, drawings in which the lines are exaggerated have been

t Strain YA 486 (area 3) which was previously reported to yield a low level of CRM, haa since been found to contain @-galactosidase in amounts slightly higher than basal levels.

40 A. V. FOWLER AND I. ZABIN

added. Plate II compares the native protein and extract,s of the two missense mut,ants,

Zl and Zu 178, and two nonsense mutants, X 90 (area 16) and NG 200 (area 11). The precipitin line of strain Z, (dimer) fuses wit,h spurring from /3-galactosidase, indicating

antigenic similarity but not identity. Strain Z, r7s (monomer) is related to Z, in a similar manner. In contrast, the precipitin line of ochre mutant X 90 fuses to Z, 178

with no spurring, indicating antigenic identity. The precipitin lines joining X 90 and

NG 200 also fuse completely. In other experiments, we have noted identical char- acteristics with extracts of all nonsense mutants mapping in areas 11 through 16.

The precipitin lines which form with ext.racts of nonsense mutants of areas 5 to 9 are more diffuse and are closer to the center well. To make the antigenic relationships

more apparent, we have used for Plate III antiserum in a dilution of 1: 4 rather than

1 :lO. From Plate III and from other observations, we found that the precipitin lines formed with extracts of these nonsense mutants, NG 125, YA 559 and U 239 (areas 6,

8 and 5) fuse with no spurring, indicating antigenic identity. There is antigenic

similarity but not identity between these shorter polypeptide chains and the longer

ones as shown by the presence of a spur at the fusion of the lines of NG 125 (area 6) and X 90 (area 16). The properties of strain NG 750 (area lo), which is not’ shown here, appear to be intermediate in immunological characteristics. Examination of the

precipitin lines formed with extracts of two deletion mutants (Plate III) gave results which are exactly those predicted from knowledge of the areas deleted in the gene: strain U 163 is antigenically identical to U 239 (area 5), and strain 2A 238 is antigen-

ically identical to X 90 (area 16).

(c) Precipitation of anti-/3-galactosidase by normal and mutant /3-galactosidase

Evidence for antigenic similarity of mutant protein and /Lgalactosidase was obtained not only by the Ouchterlony tests just described, but also by precipitation of antibody

from solution. In one experiment, 0*6,1=5 and 4.6 mg of ,%galactosidase were added to 0.25 ml. of concentrated antiserum in a final volume of 1.0 ml., and the solutions were

incubated and centrifuged as described in Materials and Methods. Only a trace of enzyme was found in the clear supernatant solutions which had contained the lower

amounts of /3-galactosidase, but about 35% of the initial enzyme activity was found in the supernatant solution of the third sample. This solution, without dilution, was

tested by double diffusion against extracts of induced nonsense mutant strains X 90, U 366, YA 559, YA 536 and U 239. No precipitin lines were formed, indicating that

anti-fi-galactosidase itself is the antibody which reacts with the prematurely termin- ated chains.

Experiments were also carried out in a reciprocal manner. Table 2 presents data obtained from a study in which anti-/3-galactosidase was treated with varying amounts

of an extract of X 90 (area 16) as precipitating antigen. Supernatant solutions were then tested for their ability to react with extracts of other mutants. It may be seen that the antibody reactions to strains X 90 and Z, 17R (missense monomer) were eliminated, but antibody to strain Z, (missense dimer) was not removed.

(d) Inhibition of /3-galactosidase precipitation by mutant protein

A numerical approximation of the antigenic similarity between mutant and native /3-galactosidase can be obtained by quantitative titrations as described by Perrin (1963). In his experiments, immunologically active proteins were purified from the two missense mutants, Z, and Z, 1,8r and were shown to be lOOo/o and about 20%

41 IMMUNOLOGY OF /?-GALACTOSIDASE MUTANTS

TABLE 2

Removal of anti-/3-galactosidme by precipitation with nonsense mutant extract

x 90 Mutant tested

x ‘30 Z” 178 Zl

CRM (units/ml. added)

0 t- t- ++ 100 t -t t+ IT 200 t i- ++ ++ 400 0 0 ++

1000 0 0 ++ 2000 0 0 ++

Extracts of X 90 were added to 0.05 ml. of concentrated antiserum and incubated in a final volume of 1 ml. After removal of the antigen-antibody precipitate, the supernatant solution was tested against an excess of each antigen (1000 to 2000 CRM units/ml.) by the Ouchterlony method. + +, and 0 refer to positive and no reaction, respectively.

cross-reacting, respectively, on an equal weight basis. We have tested crude extracts of the same mutants with essentially identical results. The standard titration (curve A in Fig. 3) was obtained by addition of increasing amounts of /3-galactosidase to a fixed quant,ity of antiserum in a series of tubes. After incubation and centrifugation, @- galactosidase remaining in the supernatant solution was determined. Interference with precipitation of the p-galactosidase-anti+-galactosidase complex by CRM results in a shift of the titration curve towards the origin, and from the extent of the shift the degree of antigenic similarity can be calculated. This is illustrated by curves

L 0 2000 4000 6000 8000

/3 - Calactosidase added (units)

FIQ. 3. Inhibition of j%galactosidase precipitation by mutant protein. Curve A, standard titration of antiserum with j%galactosidase; curve B, 0.07 mg protein of Z, added; curve C, 1.6 mg protein of X 90 added; curve D, 1.8 mg protein of ZV 178 added.

42 A. V. FOWLER AND I. ZABIN

B, C and D in Fig. 3. Extracts of the missense mutants Z, and Z, 178 both caused a marked shift of the titration curves (B and D), but approximately ten times more of

the Zu 178 extract was required. An extract of the nonsense mutant X 90 (area 16) caused a similar shift on addition of a large quantity of extract approximately equal to that of Z, 178. These experiments were carried out with extracts of induced Hfr 3000. Identical results were obtained when pure /%galactosiclase was used instead.

Application of this method to extracts of many additional nonsense mutants gave the results summarized in Table 3. The data are expressed as percentage cross-

TABLE 3

Inhibition by mutant extract of quantitative I)reci(pitation of p-galuctosidase

Mutant Al%% Cross-reaction

% Z, 100 Z ” 178 lo-15 x 90 16 5-10 U 366 13 10 NG 200 11 5-10 NG 750 10 l-2 YA 404 9 0 YA 559 8 0 YA 536 7 l-2 x 82 I l-2 NC 125 6 2-5 u 239 5 1-2 X 64 3 0

reactivity compared to lOOo/o for an extract of n&sense mutant Z,, calculated on the basis of identical protein content. In no case was the percentage of cross-reactivity higher than 10% for an extract of any nonsense mutant. It may be seen, however, that although these values are low, they agree well with the analyses obtained by the double diffusion procedure.

(e) Micro-mplement $x&ion

Nonsense mutant extracts were also tested for the presence of antigenically active material by the far more sensitive micro-complement fixation procedure of Wasserman & Levine (1961). Analyses were carried out with a fixed amount of antiserum, and the antigen is measured in terms of the number of micrograms of protein in the crude extract required to lyse one-half of the sensitized red blood cells (percentage C’F fixed). Figure 4 shows some representative data obtained with three induced strains, Hfr 3000 (wild type), X 90 (area 16) and NG 422 (area 6). The bar graph (Fig. 5) summarizes the values obtained for all the nonsense mutants. These experiments were carried out with extracts containing less than 100 pg of protein; extracts of uninduced cultures at these low levels, except for those of constitutive strains, had no immunological activity. Comparison of Figure5 with Figure2 shows a quite similar pattern. However, it is of interest to note that mutant strains YA 536 (area 7), YA 559 (area 8) and YA 404 (area 9) completely lack the ability to fix complement, although immunologi- cally active material was detected by the Ouchterlony procedure.

IMMUNOLOGY OF j3-GALACTOSIDASE MUTANTS

IOO-

0 01 0.1 I.0 IO 100

Protein in crude extract Cpg)

FIQ. 4. Micro-complement fixation. Curve A, Hfr 3000 (wild-type); curve B, X 90 (area 16): curve C, NC 422 (area 5). The concentrated antiserum was used at a 1: 10,000 dilution.

These analyses were performed using an antibody dilution of 1 :lO,OOO. When an antibody dilution of 1: 35,000 was used, similar values were obtained for mutants in areas 10 through 16, whereas larger amounts of extract from mutants in areas $6 and 7 were required to lyse one-half of the sensitized cells. Antibody dilutions of less than 1:10,000 could not be used because complement fixation occurred in the absence of antigen.

(f) &ability a7wJ inactivation

Experiments have been carried out to investigate the effect of various treatments on immunological activity of nonsense mutants. Antigen in crude extracts is stable to several cycles of freezing and thawing, and is also stable to incubation at 37°C for at least four hours. However, after 24 hours at 37”C, strain NG 125 (area 6) lost about 50% of the original CRM activity. In the same length of time, strains NG 200 (area 11) and X 90 (area 16) lost only about 10% of the activity.

“I

- 5 6 7 09

Map position of nonsense mutation (area)

FIG. 5. Micro-complement fixation analyses of nonsense mutants. A, B, C and D are strtLina aa listed in legend to Fig. 2. The concentrated antiserum wa,s used at a 1: 10,000 dilution.

44 A. V. FOWLER AND I. ZABlN

TABLE 4

Effect of heat on antigenic activity

Temperature Strain “C NG 125 NG 200 x 90 Hfr 3000

40 + + + +

45 1 + + 50 55 0

1 + : + +

57.5 0 Ik + + 60 0 0 0 0

Extracts containing 5 mg of protein/ml. were heated for 30 min and tested for antigenic activity by the Ouchterlony method. +, f and 0 refer to positive, trace and no reaction, respectively.

Stability of CRM activity to heat was studied by the experiment shown in Table 4. Crude extracts of induced cultures of strains NG 125, NG 200, X 90 and Hfr 3000 at a protein concentration of 5 mg/ml. were heated for 30 minutes at the temperatures indicated. It can be seen that the immunological activity of the shorter polypeptide chain (NG 125) is less stable to heat than that of the longer chains (NG 200 and X 90), and that the latter have approximately the same heat stability as ,B-galactosidase.

Treatment with sodium dodecyl sulfate showed a striking difference between the stability of mutant and native protein. /l-Galactosidase lost no enzymic activity nor immunological activity in 0.5% sodium dodecyl sulfate at room temperature for at least 16 hours. However, extracts of nonsense mutant strains U 239, NG 125, YA 559, NG 200 and X 90 (areas, 5, 6, 8, 11 and 16) in 0.2% sodium dodecyl sulfate lost all immunological activity when tested by the Ouchterlony procedure. The data shown in Table 5 illustrate these findings and show further the greater ease of denaturation of antigen of strain NG 125 compared to those of NG 200 and X 90.

These results indicate that a three-dimensional, rather than a random configuration, is required for the expression of immunological activity by these incomplete poly- peptide chains. Further, as would be expected, differences in stability to denaturing conditions of the various chains could be demonstrated.

TABLE 5

Effect of detergent on antigenic activity

Sodium dodecyl sulfate

(%)

Strain

NG 125 NG 200 x 90 Hfr 3000

Extracts containing 3 CRM units were mixed with sodium dodecyl sulfate and were analyzed by the Ouohterlony method. + and 0 refer to positive and no reaction, respectively.

IMMUNOLOGY OF ,!?-GALACTOSIDASE MUTANTS 45

4. Discussion

By three different immunological tests, the Ouchterlony, micro-complement fixa- tion and inhibition procedures, we have shown that incomplete polypeptide chains formed by many /I-galactosidase nonsense mutants react with antiserum prepared against the complete native protein. Quantitative analyses by the three methods of the level of antigenic activity in extracts of these mutants and in extracts of several deletion and missense mutants agree in general. It is worth noting that inhibition of titration of antibody with fi-galactosidase by addition of mutant protein requires addition of relatively large amounts of such protein; this is reminiscent of the finding by Kaplan, Ensign, Bonner & Mills (1964) that immunologically active material could be detected in extracts of certain tryptophan synthetase mutants by comple- ment fixation, but not by inhibition tests.

There can be no doubt that the antigen measured with anti-/I-galactosidase is actually the product of the Z gene. Induction of the lac operonis required (except for several constitutive strains) for a positive response to purified antiserum, and re- moval of antibody with pure ,!I-galactosidase eliminates cross-reaction. Further evidence for antigenic similarity comes from the observation that precipitin lines of nonsense mutant extracts fuse with spurring to the line produced with native fl- galactosidase.

Examination of the relationships of precipitin lines among the various mutants reveals some interesting features, and suggests the existence of three gross classes of antigenic determinants in /I-galactosidase. Two of these reside within the incomplete chains themselves. Those nonsense mutants which map in the terminal part of the gene (areas 11-16) form one class, and those nonsense mutants which map near the center of the gene (areas 5-7) form another class of antigenically reactive proteins. It has been shown previously that three of the longer chains and one of the shorter exist in these extracts in monomeric form. Though differences may very well exist within each group of antigenically reactive proteins, none can be detected by this procedure.

Studies with the three deletion mutants are exactly in accord with these suggestions. Mutant strain 2A238, in which most of the chain is present, behaves like the first group of nonsense mutants, those that form most of the polypeptide. Mutant strain U 163, in which a deletion begins just before the center of the gene, is antigenically identical to the shorter group of nonsense mutant chains. Mutant strain 4680, in which the gene is deleted from near the end of the first third through the second third of the gene, forms no CBM whatsoever. This latter mutant produces both the alpha and omega fragments which complement with appropriate extracts of /3-galactosidase mutants to produce enzymic activity (Ullmann, Jacob & Monod, 1967 ; Ullmann, Perrin, Jacob & Monod, 1965). These might represent separate subunits of the /3-galactosidase monomer, but more likely (Brown, Koorajian, Katze & Zabin, 1966; Newton, 1966) are degradative products of the mutant polypeptide produced in extracts. In any event these fragments must be devoid of immunological activity. This leads to the interesting possibility that these portions of the native /Lgalactosidase may be more or less buried within the complete molecules and hence do not evoke antibody response.

A third, less definite class of antigenic determinant may be considered to be a con- sequence of the polymeric nature of the antigen, and is derived from the fact that the line of the n&sense mutant, Z,, which forms a dimer, fuses with spurring to the ,&galactosidase line. The precipitin lines of all other mutants tested here fuse with

46 A.V. FOWLER AND I.ZABIN

spurring to the lines of /3-galactosidase and of Z,. Therefore these proteins are anti- genitally similar but not identical to the polymeric protein. (Steers & Shifrin (1967) have reported that a different /3-galactosidase-negative mutant forms a CRM which

exists in dimeric form and is immunologically identical to the native protein.) The observation that extracts of nonsense mutants of areas 11 through 16 contain

relatively high levels of antigenic activity whereas extracts of mutants of areas 1 through 4 contain no activity is explained simply by assuming that the longer chains contain antigenic determinants, but the very short chains do not. It was unexpected, however, to find that extracts of mutants of areas 8 and 9 contain minimal levels of antigenic activity. One explanation for this result is that these mutants and perhaps certain others produce polypeptide chains at lower rates. This seems unlikely, and is furthermore difficult to reconcile with the report of Zipser & Newton (1967) that deletions in the Z gene distal to the site of the nonsense codon decrease the polarity effect on transacetylase. A second explanation is that certain incomplete polypeptide chains are more subject than others to proteolytic degradation; however, our experiments show reasonable stability in extracts.

The most likely explanation would appear to be that nonsense mutants produce polypeptide chains in equimolar amounts, but that the specific immunological activi- ties of such chains vary due to different conformations. Certain of these chains may be more, and others less, like the native, perhaps by “incorrect” folding or by lack of folding. It follows that the reaction of incomplete /3-galactosidase with anti-/L galactosidase depends primarily on a particular conformation of the incomplete poly- peptide. Denaturation by heat or detergent, which eliminates antigenic reactivity, supports this interpretation.

Experiments presented elsewhere have shown that the amino-terminal sequence of fi-galactosidase is threonylmethionine, that the longer incomplete polypeptide chains (areas 11-16) contain the same sequence, but that shorter chains do not (Katze, Sridhara & Zabin, 1966 ; Brown, Brown & Zabin, 1967 ; Sridhara & Zabin, unpub- lished work). This apparent paradox may be resolved by the assumption that cleavage of the amino acid which initiates polypeptide chain synthesis in Escherichiu co&, N-formylmethionine (Capecchi, 1966), is influenced by the three-dimensional structure of the polypeptide substrate.

The existence of two antigenic classes, those of longer and of shorter incomplete chains, and their differing susceptibilities to denaturation, suggest the possibility that the two classes behave to the cleavage system as two different substrates. Thus only the longer chains are hydrolyzed at the “normal” site to expose threonylmethionine.

This work was supported in part by grant no. AI-04181 from the U.S. National Institutes of Health.

REFERENCES

Berg, A. & Zabin, I. (1964). J. Mol. Biol. 10, 289. Brown, J. L., Brown, D. M. & Zabin, I. (1967). Proc. Nat. Ad. Sci., Wash. 58, 1139. Brown, J. L., Koorajian, S., Katze, J. & Zabin, I. (1966). J. Biol. Chem. 241, 2826. Capecchi, M. R. (1966). Proc. Nat. Acad. Sci., Wash. 55, 1517. Fowler, A. & Zabin, I. (1966). Science, 154, 1027. Herzenberg, L. A. (1959). Biochim. biophys. Acta, 31, 525. Horiuchi, T., Tomizawa, J. & Novick, A. (1962). Biochim. biophye. Acta, 55, 152. Kaplan, S., Ensign, S., Bonner, D. M. & Mills, S. E. (1964). Proc. Nat. AC&. Sci., Wash. 51,

372.

IMMUNOLOGY OF ,6-GALACTOSIDASE MUTANTS 47

Karlsson, U., Koorajian, S., Zabin, I., Sjostrand, F. S. & Miller, A. (1964). J. UZt~a&uct. Be.% 10, 467.

Katze, J., Sridhara, S. & Zabin, I. (1966). J. Biol. Chem. 241, 6341. Lowry, 0. XI., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1961). J. Biol. Chem. 193,

265. Newton, A. (1966). Cold Spr. Had. Symp. Quant. Biol. 31, 81. Newton, W. A., Beckwith, J. R., Zipser, D. & Brenner, S. (1966). J. Mol. BioZ. 14, 290. Ouchterlony, 0. (1949). Acta Path. Microbial. Stand. 26, 507. Perrin, D. (1963). Ann. N.Y. Ad. Sci. 103, 1058. Steers, E., Jr. & Shifrin, S. (1967). B&him. biophys. Actu, 133, 464. Ulhnann, A., Jacob, F. & Monod, J. (1967). J. Mol. Bid. 24, 339. Ullmann, A., Perrin, D., Jacob, F. & Monod, J. (1966). J. MoZ. Biol. 12, 918. Wasserman, E. &z Levine, L. (1961). J. ImmunoE. 87, 290. Zabin, I. (1963). J. Biol. Chem. 238, 3300. Zipser, D. & Newton, A. (1967). J. Mol. Biol. 25, 567.