Vol. 28, No. 1
JOURNAL OF VIROLOGY, Oct. 1978, p. 270-278 0022-538X/78/0028-0270$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Anomalous Behavior of Bacteriophage X Polypeptides in Polyacrylamide Gels: Resolution, Identification, and Control of the X rex Gene Product MARLENE BELFORTt Department of Microbiological Chemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Received for publication 20 April 1978
The resolution of X proteins was compared on the two types of sodium dodecyl sulfate-polyacrylamide gels commonly in use. The two kinds of gel differ essentially in the ratio of the cross-linker, N'-N-bismethylene-acrylamide (bisacrylamide), to acrylamide monomer. Several A proteins migrate relatively more slowly in gels with high bisacrylamide/acrylamide ratios (HB gels) than in gels with low ratios, although the two types of gel are of roughly equivalent porosity. This effect is illustrated by a change in relative position of both the Rex and Int proteins, with apparent increases in molecular weight of about 8 and 15%, respectively, in the HB gels. This work confirms that like repressor and Int, the 28.5-kilodalton protein, identified as Rex on HB gels, is postively regulated by the XcII and cIII products and negatively controlled Cro. An intact y site is required for Rex and repressor expression after infection, whereas their synthesis in a lysogen is dependent upon a functional maintenance promoter, Pm. The X genome has the capacity to code for about 50 proteins, each containing an average of 300 amino acids. Approximately one-half of these gene products have been identified on polyacrylamide gels (13, 17, 22, 25, 27; C. Epp and M. Pearson, manuscript in preparation). In these studies, X proteins, synthesized after infection of UV-irradiated cells, have been well resolved in the 10- to 70-kilodalton (kd) size range and have lent some interesting insights into the phage's regulatory process (17, 24, 27; Epp and Pearson, in preparation). When comparing results from different laboratories, however, certain inconsistencies in relative band migration have become apparent. This is manifested in specific bands switching position relative to each other in different gel systems, in certain proteins co-migrating in one gel system but being resolved into several species in another, and in discrepancies in polypeptide molecular-weight estimates (see reference 17). In this communication, these effects are illustrated by studying the anomalous behavior of the Int and Rex proteins of X (Fig. 1) in the two gel systems commonly in use. The Int protein catalyzes the site-specific integration of X DNA into the host chromosome during lysogenization and is also involved in the excision event upon induction of the prophage (for a review, see reference 8). The Rex protein was originally defined on the basis t Present address: Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201. 270
of its property of excluding T4rII phage from infecting a X lysogen (4). Its role in the X life cycle has not yet been clearly established (see Discussion). The two types of gel used differ essentially in the ratio of cross-linker, N,N-bismethyleneacrylamide (bisacrylamide), to acrylamide monomer (5, 18). I have identified Rex on highbisacrylamide/acrylamide (HB) gels (bisacrylamide/acrylamide ratio of 8:300; 18) and show that it is obscured on low-bisacrylamide/ acrylamide (LB) gels (bisacrylamide/acrylamide ratio of 1:300; 5) since it co-migrates with repressor and since the rex gene is subject to the same regulatory controls as is the repressor gene, cI (1, 11, 21). MATERIALS AND METHODS Bacterial and bacteriophage strains. The Escherichia coli K-12 strain 159 uvr- hcr- su- (19) and its lysogenic derivative 159(Aind-), carrying a noninducible prophage, were used as hosts in infection experiments. Phage strains used carried the following mutations, singly or in various combinations: cI857 (34), cIam34, and cy42 (15); cIIam60am41 (3), cIIIam6ll (33), crol6 (26), prmll6 (37), rex5a, and rexamQ (10); and int-c226 (31), Sam7 (12), and imm43 (16). Infections. Growth of cells, irradiation, starvation, infection, and labeling with '4C-labeled mixed amino acids were performed as previously described (27). When labeled with [3S]methionine, cells were grown and labeled in minimal medium that had not been supplemented with unlabeled amino acids. Labeling was terminated by the addition of 100 jig of L-methi-
VOL. 28, 1978
ALTERED A PROTEIN RESOLUTION: THE rex PRODUCT
Pi r
0o/o
DEI Head
V I
I
20
I
Tail
b
att int1-I 60 recomb
Pl PR N imm- -OP 80 reg
rep
271
0a
WIJ% Iysis I/ys
a b I imm 434 Pi
°l
PL
0a ,Pre PR ? v
bE.a4ObEaS6 att int xis Ea22 exo bet cMIEa1l N rex cI cro clI OP
IntC C
Prm
4
d FIG. 1. Genetic and transcriptional map ofphage A. The upper map shows the general organization of the A genome and its division into regions specifying morphological functions, b-region proteins, recombination functions, regulatory controls, and the replication and lysis functions. The lower map is an expansion of the upper, running from about 45% to about 85% of A DNA. Promoter and operator sites are indicated above the maps, and cis-acting mutations used in this study are labeled below the map with arrows pointing upward to the site of the mutation. Gene products identified on the gels as well as relevant regulatory proteins have been assigned to their appropriate map positions. Arrows denoting mRNA transcripts are as follows: (a) Nstimulated early transcripts originating at the PL and PR promoters; (b) Q-dependent late transcription initiated at PR'; (c) cII/cIII-stimulated early cI-rex and int transcripts originating at Pre (exact position not known) and PI, respectively; (d) repressor-autoregulated cI-rex transcript synthesized in a lysogen from P,. All early transcripts (a and c) are shut off later in infection by the Cro product which originates from the PR message. In the establishment of lysogeny, repressor and Rex are synthesized from the cII/cIII-stimulated transcript. An intact y site on the DNA is required for this. Repressor then binds to operator sites OL and OR to silence PL and PR. Once lysogeny is established, repressor and Rex are synthesized from the message originating from promoter P,,,n which is located within OR. For a detailed account of the regulatory interplay of these elements and their influence on A's life cycle, see references 8, 14, and 29. onine per ml and concomitant chilling. Washed pellets were suspended in sampling buffer containing sodium dodecyl sulfate (SDS) and mercaptoethanol and stored at -20°C until electrophoresis (see reference 27).
Electrophoresis and autoradiography. Samples were boiled for 3 min and centrifuged to pellet insoluble material, and 10-/A samples were loaded into wells of the spacer gel. The 20% acrylamide LB gels were run at 20 mA/gel for 8 to 9 h. The 10 to 15% acrylamide HB gels were run at 18 mA/gel for 5 to 6 h. Running buffer used was the same for both systems and contained 6 g of Tris and 28.8 g of glycine per liter of 0.1% SDS solution. The composition of the different gels is given in Table 1. Staining and fixation. To stain unlabeled proteins, gels were soaked for 1 h in a solution containing 0.2% Coomassie brilliant blue, 50% trichloroacetic acid, and 7% methanol. Destaining was achieved by gently agitating the gels for 24 h in about 10 volumes of destaining solution (5% acetic acid, 7% methanol). Destaining solution was changed two to three times during the destaining period. When staining was not
necessary, gels were fixed in 50% trichloroacetic acid and rinsed in destaining solution. Sometimes gels were dried directly after electrophoresis to avoid washing out low-molecular-weight polypeptides during the rinsing process. This accounts for darkly shaded areas in the lower section of some gels (see Fig. 6). Analysis of autoradiograms. After drying, gels were exposed to no-screen Kodak X-ray film for 4 and 14 days when labeling was with 14C and for 1 and 4 days when 35S label was used. The longer exposures were photographed in each case, whereas the films which had been exposed for shorter times were analyzed by scanning the bands with a Joyce-Loebl microdensitometer.
RESULTS Resolution of X proteins on polyacrylamide gels of different composition. When electrophoretic mobility was plotted against the logarithm of the known molecular weight of protein standards, relatively smooth curves were obtained down to about 20 kd in the two gel
272
BELFORT
J.- VIROL.
TABLE 1. Composition of separating gelsa Final concn
Component LB gels HB gels Acrylamide 20% 15% 12.6% Bisacrylamide 0.067% 0.4% 0.336% Tris-hydrochloride pH 7.8 0.26 M pH 8.8 0.375 M 0.375 M SDS 0.1% 0.1% 0.1% TEMEDb 0.03% 0.03% 0.03% Ammonium persulfate 0.03% 0.03% 0.03% a The 20% LB separating gel (5) has a ratio of bisacrylamide to acrylamide of 1:300, whereas the bisacrylamide/acrylamide ratio of HB gels (18) is 8:300. Spacer gels are similar in the two systems and contain 6% acrylamide, 0.15% bisacrylamide, 0.15 M Tris-hydrochloride, pH 6.8, 0.1% SDS, 0.06% TEMED, and 0.06% ammonium persulfate. N,N,N',N-tetramethylethylenediamine. Its final concentration in the gels is stated on a volume/volume percent basis.
systems being studied (Fig. 2). Anomalies started to appear in the molecular-weight range below this, and this study is therefore confined to those A proteins of higher molecular weight. Interestingly, whereas most X proteins above 20 kd exhibited similar electrophoretic migration behavior in the LB and HB gel systems, several polypeptides migrated relatively more slowly in the HB gels, resulting in increased molecularweight estimates in this system (Fig. 2 and 3). This is true for the bEa56, Int, Rex, P, and Ea22 proteins of A. In Fig. 2, proteins bEa56, Int, Rex, and Ea22 correspond to numbers 1, 3, 7, and 10, respectively. The bEa56, P, and Ea22 bands in Fig. 3 are numbered 2, 8, and 11, respectively, whereas Int and Rex are labeled as such. Since the kinds of artifacts which arise as a result of altered protein mobility in different gel systems are well illustrated for the cases of Int and Rex, and since the regulation of these proteins is of interest, we will restrict our attention to the migratory behavior of these two proteins: Int migrated ahead of E (38 kd) with an apparent mass of 36 kd in the 20% LB gel, whereas it migrated behind E with an apparent mass of 41 kd in the HB gels (Fig. 2-6). Rex, on the other hand, was completely obscured in the 20% LB gels, since it co-migrated with repressor at 26 kd. In the HB gels, however, Rex was clearly resolved into a more slowly migrating species with an apparent mass of 28.5 kd (see below). Electrophoretic behavior of X proteins expressed in a lysogen. Upon electrophoresis of extracts of lysogens infected with AcI857S7 on LB and HB gels, the following paradox emerged: only one band, at 26 kd, was apparent in a 20% LB gel (Fig. 4b), whereas two bands were resolved in the HB system, the second band migrating at 28.5 kd (Fig. 4b, 51). From infections with amber mutants defective in genes cI and
rex, the two genes known to be expressed in a lysogen (28), it becomes clear that the 26-kd band seen in the 20% LB gels was in fact a composite band, comprising repressor and Rex. In HB gels, the 26-kd band was absent upon infection with an amber mutant defective in the cI gene (Fig. 4d) and corresponded to repressor, whereas the 28.5-kd band appeared in a rex missense mutant infection (Fig. 4c, 5n), but not
bc
a
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2
40I ~~~~30~~~~~~
°-
w6
-J~~~~7
(-
w
20
0 1:
0
,
-
"
% -i 0-0'
10
0
I
'.
0.2 0.4 0.6 0.8 MOBILITY (RF)
X
1.0
FIG. 2. Mobilities of A-directed proteins in gels of different composition. Labeled A proteins and unlabeled protein standards were electrophoresed on HB gels with a bisacrylamide/acrylamide ratio of 8:300 (a) and (b), and their migration was compared with that on an LB gel with a bisacrylamide/acrylamide ratio of 1:300 (c). The acrylamide concentrations were 15, 12.6, and 20% for (a), (b), and (c), respectively. After electrophoresis, gels were stained, dried, and autoradiographed as described in Materials and Methods. The plots were calibrated by using the following protein standards (0) with mass given in parentheses in kilodaltons: (I) bovine serum albumin (68); (II) catalase (60); (III) immunoglobulin, H chain (50); (IV) ovalbumin (43); (V) immunoglobulin L chain (23.5); (VI) trypsin (23.3); (VII) lysozyme (14.3); (VIII) chymotrypsin, H chain (13); (IX) cytochrome c (11.7); and (X) chymotrypsin, L chain (11). The closed circles represent phage-directed proteins: (1) bEa56; (2) bEa4O; (3) Int; (4) E; (5) V; (6) bet + bEa3O; (7) Rex; (8) repressor; (9) exo; (10) Ea22. Data for (c) are taken from reference 27. The reader should note that the numbering system for specific proteins differs from figure to figure.
ALTERED A PROTEIN RESOLUTION: THE rex PRODUCT
VOL. 28, 1978
b
a
273
d
c
1+ 2
; --->Jnt
2% -
n \-
l
-
.
=
-~~~--_4='..
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: 6
JOURNAL OF VIROLOGY, Oct. 1978, p. 270-278 0022-538X/78/0028-0270$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Anomalous Behavior of Bacteriophage X Polypeptides in Polyacrylamide Gels: Resolution, Identification, and Control of the X rex Gene Product MARLENE BELFORTt Department of Microbiological Chemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Received for publication 20 April 1978
The resolution of X proteins was compared on the two types of sodium dodecyl sulfate-polyacrylamide gels commonly in use. The two kinds of gel differ essentially in the ratio of the cross-linker, N'-N-bismethylene-acrylamide (bisacrylamide), to acrylamide monomer. Several A proteins migrate relatively more slowly in gels with high bisacrylamide/acrylamide ratios (HB gels) than in gels with low ratios, although the two types of gel are of roughly equivalent porosity. This effect is illustrated by a change in relative position of both the Rex and Int proteins, with apparent increases in molecular weight of about 8 and 15%, respectively, in the HB gels. This work confirms that like repressor and Int, the 28.5-kilodalton protein, identified as Rex on HB gels, is postively regulated by the XcII and cIII products and negatively controlled Cro. An intact y site is required for Rex and repressor expression after infection, whereas their synthesis in a lysogen is dependent upon a functional maintenance promoter, Pm. The X genome has the capacity to code for about 50 proteins, each containing an average of 300 amino acids. Approximately one-half of these gene products have been identified on polyacrylamide gels (13, 17, 22, 25, 27; C. Epp and M. Pearson, manuscript in preparation). In these studies, X proteins, synthesized after infection of UV-irradiated cells, have been well resolved in the 10- to 70-kilodalton (kd) size range and have lent some interesting insights into the phage's regulatory process (17, 24, 27; Epp and Pearson, in preparation). When comparing results from different laboratories, however, certain inconsistencies in relative band migration have become apparent. This is manifested in specific bands switching position relative to each other in different gel systems, in certain proteins co-migrating in one gel system but being resolved into several species in another, and in discrepancies in polypeptide molecular-weight estimates (see reference 17). In this communication, these effects are illustrated by studying the anomalous behavior of the Int and Rex proteins of X (Fig. 1) in the two gel systems commonly in use. The Int protein catalyzes the site-specific integration of X DNA into the host chromosome during lysogenization and is also involved in the excision event upon induction of the prophage (for a review, see reference 8). The Rex protein was originally defined on the basis t Present address: Division of Laboratories and Research, New York State Department of Health, Albany, NY 12201. 270
of its property of excluding T4rII phage from infecting a X lysogen (4). Its role in the X life cycle has not yet been clearly established (see Discussion). The two types of gel used differ essentially in the ratio of cross-linker, N,N-bismethyleneacrylamide (bisacrylamide), to acrylamide monomer (5, 18). I have identified Rex on highbisacrylamide/acrylamide (HB) gels (bisacrylamide/acrylamide ratio of 8:300; 18) and show that it is obscured on low-bisacrylamide/ acrylamide (LB) gels (bisacrylamide/acrylamide ratio of 1:300; 5) since it co-migrates with repressor and since the rex gene is subject to the same regulatory controls as is the repressor gene, cI (1, 11, 21). MATERIALS AND METHODS Bacterial and bacteriophage strains. The Escherichia coli K-12 strain 159 uvr- hcr- su- (19) and its lysogenic derivative 159(Aind-), carrying a noninducible prophage, were used as hosts in infection experiments. Phage strains used carried the following mutations, singly or in various combinations: cI857 (34), cIam34, and cy42 (15); cIIam60am41 (3), cIIIam6ll (33), crol6 (26), prmll6 (37), rex5a, and rexamQ (10); and int-c226 (31), Sam7 (12), and imm43 (16). Infections. Growth of cells, irradiation, starvation, infection, and labeling with '4C-labeled mixed amino acids were performed as previously described (27). When labeled with [3S]methionine, cells were grown and labeled in minimal medium that had not been supplemented with unlabeled amino acids. Labeling was terminated by the addition of 100 jig of L-methi-
VOL. 28, 1978
ALTERED A PROTEIN RESOLUTION: THE rex PRODUCT
Pi r
0o/o
DEI Head
V I
I
20
I
Tail
b
att int1-I 60 recomb
Pl PR N imm- -OP 80 reg
rep
271
0a
WIJ% Iysis I/ys
a b I imm 434 Pi
°l
PL
0a ,Pre PR ? v
bE.a4ObEaS6 att int xis Ea22 exo bet cMIEa1l N rex cI cro clI OP
IntC C
Prm
4
d FIG. 1. Genetic and transcriptional map ofphage A. The upper map shows the general organization of the A genome and its division into regions specifying morphological functions, b-region proteins, recombination functions, regulatory controls, and the replication and lysis functions. The lower map is an expansion of the upper, running from about 45% to about 85% of A DNA. Promoter and operator sites are indicated above the maps, and cis-acting mutations used in this study are labeled below the map with arrows pointing upward to the site of the mutation. Gene products identified on the gels as well as relevant regulatory proteins have been assigned to their appropriate map positions. Arrows denoting mRNA transcripts are as follows: (a) Nstimulated early transcripts originating at the PL and PR promoters; (b) Q-dependent late transcription initiated at PR'; (c) cII/cIII-stimulated early cI-rex and int transcripts originating at Pre (exact position not known) and PI, respectively; (d) repressor-autoregulated cI-rex transcript synthesized in a lysogen from P,. All early transcripts (a and c) are shut off later in infection by the Cro product which originates from the PR message. In the establishment of lysogeny, repressor and Rex are synthesized from the cII/cIII-stimulated transcript. An intact y site on the DNA is required for this. Repressor then binds to operator sites OL and OR to silence PL and PR. Once lysogeny is established, repressor and Rex are synthesized from the message originating from promoter P,,,n which is located within OR. For a detailed account of the regulatory interplay of these elements and their influence on A's life cycle, see references 8, 14, and 29. onine per ml and concomitant chilling. Washed pellets were suspended in sampling buffer containing sodium dodecyl sulfate (SDS) and mercaptoethanol and stored at -20°C until electrophoresis (see reference 27).
Electrophoresis and autoradiography. Samples were boiled for 3 min and centrifuged to pellet insoluble material, and 10-/A samples were loaded into wells of the spacer gel. The 20% acrylamide LB gels were run at 20 mA/gel for 8 to 9 h. The 10 to 15% acrylamide HB gels were run at 18 mA/gel for 5 to 6 h. Running buffer used was the same for both systems and contained 6 g of Tris and 28.8 g of glycine per liter of 0.1% SDS solution. The composition of the different gels is given in Table 1. Staining and fixation. To stain unlabeled proteins, gels were soaked for 1 h in a solution containing 0.2% Coomassie brilliant blue, 50% trichloroacetic acid, and 7% methanol. Destaining was achieved by gently agitating the gels for 24 h in about 10 volumes of destaining solution (5% acetic acid, 7% methanol). Destaining solution was changed two to three times during the destaining period. When staining was not
necessary, gels were fixed in 50% trichloroacetic acid and rinsed in destaining solution. Sometimes gels were dried directly after electrophoresis to avoid washing out low-molecular-weight polypeptides during the rinsing process. This accounts for darkly shaded areas in the lower section of some gels (see Fig. 6). Analysis of autoradiograms. After drying, gels were exposed to no-screen Kodak X-ray film for 4 and 14 days when labeling was with 14C and for 1 and 4 days when 35S label was used. The longer exposures were photographed in each case, whereas the films which had been exposed for shorter times were analyzed by scanning the bands with a Joyce-Loebl microdensitometer.
RESULTS Resolution of X proteins on polyacrylamide gels of different composition. When electrophoretic mobility was plotted against the logarithm of the known molecular weight of protein standards, relatively smooth curves were obtained down to about 20 kd in the two gel
272
BELFORT
J.- VIROL.
TABLE 1. Composition of separating gelsa Final concn
Component LB gels HB gels Acrylamide 20% 15% 12.6% Bisacrylamide 0.067% 0.4% 0.336% Tris-hydrochloride pH 7.8 0.26 M pH 8.8 0.375 M 0.375 M SDS 0.1% 0.1% 0.1% TEMEDb 0.03% 0.03% 0.03% Ammonium persulfate 0.03% 0.03% 0.03% a The 20% LB separating gel (5) has a ratio of bisacrylamide to acrylamide of 1:300, whereas the bisacrylamide/acrylamide ratio of HB gels (18) is 8:300. Spacer gels are similar in the two systems and contain 6% acrylamide, 0.15% bisacrylamide, 0.15 M Tris-hydrochloride, pH 6.8, 0.1% SDS, 0.06% TEMED, and 0.06% ammonium persulfate. N,N,N',N-tetramethylethylenediamine. Its final concentration in the gels is stated on a volume/volume percent basis.
systems being studied (Fig. 2). Anomalies started to appear in the molecular-weight range below this, and this study is therefore confined to those A proteins of higher molecular weight. Interestingly, whereas most X proteins above 20 kd exhibited similar electrophoretic migration behavior in the LB and HB gel systems, several polypeptides migrated relatively more slowly in the HB gels, resulting in increased molecularweight estimates in this system (Fig. 2 and 3). This is true for the bEa56, Int, Rex, P, and Ea22 proteins of A. In Fig. 2, proteins bEa56, Int, Rex, and Ea22 correspond to numbers 1, 3, 7, and 10, respectively. The bEa56, P, and Ea22 bands in Fig. 3 are numbered 2, 8, and 11, respectively, whereas Int and Rex are labeled as such. Since the kinds of artifacts which arise as a result of altered protein mobility in different gel systems are well illustrated for the cases of Int and Rex, and since the regulation of these proteins is of interest, we will restrict our attention to the migratory behavior of these two proteins: Int migrated ahead of E (38 kd) with an apparent mass of 36 kd in the 20% LB gel, whereas it migrated behind E with an apparent mass of 41 kd in the HB gels (Fig. 2-6). Rex, on the other hand, was completely obscured in the 20% LB gels, since it co-migrated with repressor at 26 kd. In the HB gels, however, Rex was clearly resolved into a more slowly migrating species with an apparent mass of 28.5 kd (see below). Electrophoretic behavior of X proteins expressed in a lysogen. Upon electrophoresis of extracts of lysogens infected with AcI857S7 on LB and HB gels, the following paradox emerged: only one band, at 26 kd, was apparent in a 20% LB gel (Fig. 4b), whereas two bands were resolved in the HB system, the second band migrating at 28.5 kd (Fig. 4b, 51). From infections with amber mutants defective in genes cI and
rex, the two genes known to be expressed in a lysogen (28), it becomes clear that the 26-kd band seen in the 20% LB gels was in fact a composite band, comprising repressor and Rex. In HB gels, the 26-kd band was absent upon infection with an amber mutant defective in the cI gene (Fig. 4d) and corresponded to repressor, whereas the 28.5-kd band appeared in a rex missense mutant infection (Fig. 4c, 5n), but not
bc
a
*"70 \o -A_ \ \\ 60 _U _°_ 3._ -:~~~~T1 50- 1n -
2
40I ~~~~30~~~~~~
°-
w6
-J~~~~7
(-
w
20
0 1:
0
,
-
"
% -i 0-0'
10
0
I
'.
0.2 0.4 0.6 0.8 MOBILITY (RF)
X
1.0
FIG. 2. Mobilities of A-directed proteins in gels of different composition. Labeled A proteins and unlabeled protein standards were electrophoresed on HB gels with a bisacrylamide/acrylamide ratio of 8:300 (a) and (b), and their migration was compared with that on an LB gel with a bisacrylamide/acrylamide ratio of 1:300 (c). The acrylamide concentrations were 15, 12.6, and 20% for (a), (b), and (c), respectively. After electrophoresis, gels were stained, dried, and autoradiographed as described in Materials and Methods. The plots were calibrated by using the following protein standards (0) with mass given in parentheses in kilodaltons: (I) bovine serum albumin (68); (II) catalase (60); (III) immunoglobulin, H chain (50); (IV) ovalbumin (43); (V) immunoglobulin L chain (23.5); (VI) trypsin (23.3); (VII) lysozyme (14.3); (VIII) chymotrypsin, H chain (13); (IX) cytochrome c (11.7); and (X) chymotrypsin, L chain (11). The closed circles represent phage-directed proteins: (1) bEa56; (2) bEa4O; (3) Int; (4) E; (5) V; (6) bet + bEa3O; (7) Rex; (8) repressor; (9) exo; (10) Ea22. Data for (c) are taken from reference 27. The reader should note that the numbering system for specific proteins differs from figure to figure.
ALTERED A PROTEIN RESOLUTION: THE rex PRODUCT
VOL. 28, 1978
b
a
273
d
c
1+ 2
; --->Jnt
2% -
n \-
l
-
.
=
-~~~--_4='..
In+\
: 6
