VIROLOGY

69, 617-625 (1976)

Tryptic

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Analysis of Adenovirus Binding Proteins

BRIGITTE ROSENWIRTH,’

CARL ANDERSON,2

Types 2, 5 and 12 DNA-

AND

ARNOLD J. LEVINE

Princeton University, Department of Biochemical Sciences, Princeton, New Jersey 08540, and Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 1172% Accepted August 26,1975 Each of types 2, 5 and 12 human adenovirus (Ad) induces the synthesis of singlestrand-specific DNA-binding proteins at early times after infection of human or monkey cells. The molecular weights of these proteins are: Ad2, 72,000; Ad5, 72,900; and Ad12, 58,000. In addition, each of these viruses produces a collection of two or three lower molecular weight (41,000-47,000), single-strand-specific DNA-binding proteins that appear in variable amounts after infection. Tryptic fingerprints of the peptides derived from [35S1methionine-labeled 72,000-MW (Ad2 and 5), 58,000-MW (Ad121 and 41,00047,000-MW (Ad2,5 and 12) DNA-binding proteins have been obtained. The results of this analysis permit the following conclusions: (i) The 72,000-MW (Ad2 and 5) and 58,000-MW (Ad12) proteins synthesized in adenovirus-infected cells are distinct from a 72,000-MW cellular DNA-binding protein found in uninfected or mock-infected cells. (ii) The [35S1methionine-labeled peptides derived from the 41,000-47,000-MW proteins (Ad2, 5 and 12) are similar if not identical to the [?S]methionine-labeled tryptic peptides derived from the larger (Ad2 and 5, 72,000-MW; Ad12, 58,000-MW) DNA-binding proteins. (iii) The [35Slmethionine-labeled peptides derived from the adenovirus types 2 and 5, 72,000MW proteins are quite similar but distinct from the adenovirus type 12, 58,000-MW protein. (iv) The peptide fingerprints of the adenovirus type 2, 72,000-MW protein synthesized in either human or monkey cells are very similar or identical. INTRODUCTION

Infection of human or monkey cells with the human adenovirus (Ad) type 2, 5 or 12 results in the synthesis of viral-specific proteins that bind preferentially to singlestranded DNA (van der Vliet and Levine, 1972; Rosenwirth et al., 1975). Type 2 or 5 adenovirus-infected cells each produce at least two DNA-binding proteins with molecular weights of 72,000 and 45,500, while type 12 adenovirus-infected cells contain similar DNA-binding proteins with molecular weights of 58,000 and 47,000. Temperature-sensitive mutants of type 5 adenovirus (H5ts125) and type 12 adenovirus (HlBtsA) fail to produce detectable levels of these DNA-binding proteins at the nonI Present address: Institute logne, Germany. * Present address: Brookhaven tory, Brookhaven, N. Y.

for

Virology,

National

Co-

Labora-

permissive temperature (van der Vliet et al., 1975; Rosenwirth et al., 1975). The H5tsl25-infected cells produce 72,000- and 45,500-MW DNA-binding proteins at 32” that are both thermolabile for continued binding to single-stranded DNA when compared to the wild-type proteins (van der Vliet et al., 1975). These experiments suggest that the adenovirus temperature-sensitive mutants H5ts125 and HlXtsA either code for or regulate the levels of both the large molecular weight class (72,000 and 58,000) and small molecular weight (45,000-47,000) DNA-binding proteins. There are several explanations for the observation that a temperature-sensitive mutant (H5ts125 and HlBtsA) could code for or regulate the level of two distinct molecular weight classes of proteins (72,000 or 58,000 and 45,000-47,000) in an infected cell. (i) The 45,000-47,000-MW proteins might be proteolytic breakdown prod-

617 Copyright Q 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ucts of the larger DNA-binding protein, and both proteins would then be coded for by the same gene. (ii) Both the H&125 and Hl2tsA mutants might be double mutants. (iii) The viral gene functions impaired by the H5ts125 and Hl2tsA mutations could regulate the synthesis or production of both the large and small classes of DNAbinding proteins. This last hypothesis is not however compatible with the observation that in H5tsl25-infected cells, both the 72,000- and 45,500-MW proteins are themselves thermolabile for continued binding to DNA (van der Vliet et al., 1975). One way to distinguish between these ideas is to obtain a tryptic peptide fingerprint pattern for each of these DNA-binding proteins. If the 45,000-47,000-MW proteins were indeed proteolytic breakdown products of the larger DNA-binding proteins, they should contain, at least, a subset of tryptic peptides found in the larger protein. The experiments presented in this paper demonstrate that this is indeed the case. MATERIALS

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METHODS

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tures were labeled in a similar fashion after first concentrating the cells to l-2 x lo6 cells/ml. The cells were collected and washed three times with phosphatebuffered saline. The cell pellets were resuspended in one-fifth volume of hypotonic buffer A (0.02 M Tris-HCl, pH 7.6; 0.01 M NaCl; 1.5 mit4 MgCl,; and 2.0 n-&f dithiothreitol) and frozen at -20” (in some cases stored) and thawed. Cellular debris was centrifuged out of suspension for 20 min at 15,000 g and the supernatant fluid, containing the DNA-binding proteins (van der Vliet et al., 19751, was adjusted to a final concentration of 10% glycerol (lo%, w/v), 5 mM EDTA and 15 mit4 NaCl (buffer B). This solution was applied to a single-stranded calf thymus DNA-cellulose column and the proteins were eluted as described previously (Rosenwirth et al ., 1975). All procedures were performed at 4”, and the flow rate of the DNA-cellulose column was maintained at 2 ml/hr. Sodium dodecyl sulfate (SDSLpolyacrylamide-gel electrophoresis. Electropho-

resis was performed in slab gels as described previously (Anderson et al., 1973). After electrophoresis the gels were stained with 0.25% Coomassie brilliant blue (in 5% methanol and 7.5% acetic acid). Gels were dried under vacuum and autoradiographed ([35Slmethionine-labeled proteins) with Kodak single-sided medical Xray film (SB54T). Tryptic fingerprint analysis. Peptide maps of [35Slmethionine-labeled proteins were obtained as described previously by Anderson et al. (1973).

Viruses and cell cultures. Adenovirus types 2 and 5 were grown in KB suspension cultures or monolayers on petri dishes as described (Anderson et al ., 1973; Rosenwirth et al., 1975). Adenovirus type 12 stocks were obtained in human embryonic kidney (HEK) cells (Shiroki et al., 1972). African green monkey kidney cells (AGMK) were grown in Dulbecco’s modified Eagle’s medium (DME) with 10% calf serum, penicillin, streptomycin and 2 mM glutamine (Levine et al., 1970). These cells RESULTS were infected with either adenovirus type 2, 5 or 12 at approximately 20-50 PFU Pro(Ad2 or 5) or 2-10 PFU (Ad12) per cell in Isolation ofAdenovirus DNA-Binding teins the same medium containing 2% calf seSuspension cultures of KB cells and monrum. Mock-infected cells were treated in this same way in the absence of crude viral olayer cultures of AGMK cells were infected with human adenovirus types 2,5 or extracts. Isolation of the DNA-binding proteins. 12. These cultures were labeled with Adenovirus-infected cell monolayers of [35Slmethionine from 14 to 20 hours after AGMK cells, at 37”, were labeled from 14- infection. Mock-infected monolayer cul20 hr postinfection with [35S]methionine tures of AGMK cells were labeled in the (200 &i/ml; 3.0 ml of DME containing 1.5 same fashion. The cells from each of these @/ml of methionine and 10% calf serum cultures were harvested and washed, and proteins per lo-cm plate). KB cell suspension cul- the adenovirus DNA-binding

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were prepared and isolated as described in Materials and Methods. The adenovirus DNA-binding proteins were eluted from single-stranded DNAcellulose columns in the 0.5 and 1.0 M NaCl washes (van der Vliet and Levine, 1972; Rosenwirth et al., 1975). The 1 M eluates of the DNA-cellulose columns from AdB-KB, Ad5-KB, AdB-AGMK, Ad5AGMK, AdlZ-AGMK, and mock-infected AGMK were each concentrated and further fractionated by preparative SDS-polyacrylamide-gel electrophoresis. Figure 1 shows the [3%lmethionine-labeled proteins (autoradiogram) present in these 1 M NaCl eluted fractions. [35S]methionine-labeled purified Ad2 virions (boiled in SDS)

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were also included in this gel electrophoresis to act as molecular weight standards (the virion proteins are presented on the right side of each gel in the figure). The major labeled protein species in adenovirus types 2- and &infected KB or AGMK cells was the 72,000-MW DNA-binding protein. This protein does not correspond to any detectable virion protein (van der Vliet and Levine, 1972). In addition to this major protein, several minor bands were observed in the 37,000-47,000-MW region (labeled A, B, C) of these polyacrylamide gels. With adenovirus type 1Zinfected AGMK cells, the molecular weight of the major adenovirus DNA binding protein was 58,000-60,000, as described previously

FIG. 1. Autoradiograms of preparative SDS-polyacrylamide slab-gel electrophoresis of DNA-binding proteins from adenovirus-infected cells. [YS]methionine-labeled DNA-binding proteins were prepared as described in Materials and Methods and dissolved by boiling in SDS-sample buffer. Appropriate aliquots (up to ca. 206 pg) were applied to a 120-mm-wide well in the stacking gel of a 12.5% polyacrylamide, 0.2% bisacrylamide gel prepared as described by Anderson et al. (1973). A small sample of [35Slmethionine-labeled adenovirus 2 virions, disrupted in SDS-sample buffer, was applied to a separate analytical well at the side of the same gel to provide molecular weight markers. After electrophoresis, each preparative gel was stained with Coomasaie blue for approximately 30 min and then destained overnight in 7% acetic acid, 5% methanol. Each gel was dried under vacuum at ca. 90” and autoradiographed. Bands of interest were located with the help of the stained pattern and cut out, and the polypeptides were eluted electrophoretically. Shown are portions of the autoradiograms of six preparative gels to which had been applied the 1.0 M eluant from DNA-cellulose columns on which were chromatographed (from left to right) extracts of: cl), Ads-infected KB cells labeled 14-20 hr postinfection (p.i.1; (21, Ad&infected KB cells labeled 14-20 hr p.i.; (31, Ad2infected AGMK cells labeled 14-20 hr p.i.; (4), Ad5-infected AGMK cells labeled 14-20 hr p.i.; (51, Ad12infected AGMK cells labeled 14-20 hr p.i.; and (6), mock-infected AGMK cells labeled for 6 hr. The legend on the right side of the figure indicates the positions of the major virion components as well as the major DNAbinding components found in AdlB-infected cells (60K, A’, and B’). The positions of the Ad2 and Ad5 DNAbinding components, 72K, A, B, and C, are indicated on the left.

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(Kosenwirth et al., 1975). Two minor bands were also detected in the 45,000-47,000MW region of this gel. In the mock-infected AGMK cells (and also in the mockinfected KB cells, not shown in Fig. 1) two faint bands of [35S]methionine-labeled, single-stranded DNA-binding proteins, were observed with molecular weights of approximately 72,000 and 41,000. Accurate molecular weights for these proteins were obtained from a calibration curve (Fig. 2)

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with the type 2 adenovirus virion proteins as molecular weight standards. The observation that adenovirus types 2 and 5 synthesized 72,000-MW DNA-binding proteins (in KB and AGMK cell cultures) while adenovirus type 12 synthesized a 58,000-MW DNA-binding protein in these same cells (KB cells not presented in Fig. 1) suggested that the nature of the single-stranded DNA-binding protein synthesized was indeed dependent upon the virus type employed. The presence however of two DNA-binding proteins of 72,000 and 48,000 MW in mock-infected or uninfected AGMK cells (and KB cells) brought up the possibility that the adenovirus DNA-binding proteins were cellular proteins whose synthesis was stimulated after viral infection. To determine if the adenovir-us DNA-binding proteins produced in infected cells were similar or distinct from the proteins found in uninfected cells, peptide maps of all these proteins were obtained.

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Tryptic Peptide Maps of 35S-Labeled DNABinding Proteins

lC!ii-’ cm

FIG. 2. Molecular weight determinations of adenovirus DNA-binding proteins. A graphic representation of the molecular weight vs. the electrophoretic mobility in sodium dodecyl sulfate-containing polyacrylamide gels. (A), Adenovirus type 2 DNA-binding proteins; (B), adenovirus type 12 DNA-binding proteins. Molecular weight references (as in Fig. 1) are virion proteins, II, 120,000; III, 85,000; IV, 62,000; V, 48,500; VI, 27,000; and VII, 18,500, each plotted as a separate point (01. It should be noted that, previously, the adenovirus type 5 41,000- and 45,500-MW proteins were referred to collectively as 48,000-MW proteins (van der Vliet and Levine, 1972) and the adenovirus type 12 58,000MW protein was referred to as a 60,000-MW protein (Rosenwirth et al., 1975).

To compare the major DNA-binding proteins found in adenovirus type 2-infected AGMK cells, adenovirus type 5-infected AGMK cells and mock-infected AGMK cells, the 1 M NaCl eluate of a DNAcellulose column (see Fig. 1 for the proteins analyzed) was concentrated, digested with trypsin and the [35S]methionine-labeled peptides displayed in two-dimensional fingerprints or maps (Anderson et al., 1973). The resulting autoradiogram maps of these three fractions are presented in Fig. 3. The major [35S]methionine-labeled tryptic peptides from adenovirus type 2- and 5-infected cells (1 M NaCl eluate) were similar but not identical. Both of these tryptic peptide maps however were distinct from that obtained from the mock-infected AGMK cells (1 M NaCl eluate) . In order to obtain a finer analysis of the [35Slmethionine-labeled peptides of these DNA-binding proteins, the adenovirus types 2 and 5 72,000-MW proteins synthesized in KB cells and AGMK cells were cut out and eluted from the polyacrylamide-

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FIG. 3. Tryptic fingerprint of the 1.0 M eluant from DNA-cellulose columns on which were chromatographed extracts of (A), AdS-infected AGMK cells; (B), Ad5-infected AGMK cells; (0, mock-infected AGMK cells. [%]methionine-labeled extracts were prepared and chromatographed as described in Materials and Methods and the legend to Fig. 1. Aliquots of the 1.0 M eluants were oxidized and digested with trypsin as described in Materials and Methods, and the tryptic peptides were separated on thin-layer cellulose plates by chromatography in n-butano1(3001:pyridine(6O):acetic acid(2OO):water(240) and then by electrophoresis at pH 3.5 (1000 V for 35 mini as described by Anderson et al. (1973). Methionine-containing peptides were detected by autoradiography.

gel slices (in Fig. 1). The tryptic peptide analysis of these proteins is presented in Fig. 4. The 72,000-MW DNA-binding proteins from adenovirus type 2-infected KB cells and AGMK cells yielded similar or identical P5S]methionine-labeled tryptic peptides. Similarly, the peptide maps of the type 5 adenovirus 72,000-MW DNAbinding protein were identical, independent of the cell type (KB or AGMK) in which they were synthesized. The Relationship between the 72,000-MW DNA-Binding Proteins and the Smaller Molecular Weight Proteins Figure 5 presents the tryptic peptide maps of the [35Slmethionine-labeled adenovirus type 2 72,000-MW protein (Figure 50, adenovirus type 2 45,500-MW protein (Fig. 5D, obtained from Fig. 1, band A) and adenovirus type 2 41,000-MW protein (Fig. 5E, obtained from Fig. 1, band B). The major [35Slmethionine-containing peptides are very similar in all three cases. Figure 5 also presents the fingerprint of the mock-infected KB cell 72,000-MW (Fig. 5A) and 41,000-MW proteins (Fig. 5B) which are distinct from the adenovirus type 2 72,000- and 41,000-MW peptide maps. One can see however, in the autora-

diogram of the adenovirus type 2-infected cell 41,000-MW protein (Fig. 5E), faint [35Slmethionine-peptide spots that are derived from this mock-infected KB cell 41,000-MW protein (compare Figs. 5B and E). This is expected since the adenovirus type 2- and mock-infected 41,000-MW proteins corn&rate in the SDS-polyacrylamide gel. Finally the [35Slmethionine-containing peptides from the adenovirus type 2-infected KB cell 37,000-MW protein band (Fig. 5F, obtained from Fig. 1, band C) are totally unrelated to the peptides derived from the adenovirus type 2-infected cell 72,000-, 45,500- or 41,000-MW proteins. This last analysis was performed to eliminate the possibility that the 72,000-MW protein generated proteolytic breakdown products that were distributed throughout the lower molecular weight regions of the SDS-polyacrylamide gel and therefore might be detected in every band tested in this gel (Fig. 1). Figure 6 presents the two-dimensional analysis of the [3%Imethionine-containing peptides from adenovirus type 12-infected cell 58,000-MW protein and the 47,000- and 45,000-MW proteins (labeled A’ and B’ in Fig. 1). Once again, similar [35S]methionine-peptide spots can be readily detected

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FIG. 4. Tryptic fingerprints of Ad2 and Ad5 72,000-MW binding proteins synthesized in AGMK and KB cells. [35S1methionine-labeled DNA-binding protein was prepared as described in Materials and Methods and purified by preparative SDS-polyacrylamide-gel electrophoresis as described in Fig. 1. The eluted 72,000-MW region was processed for tryptic fingerprinting as described in Materials and Methods and the legend to Fig. 3. Shown are the fingerprints (autoradiogramsl of the 72,000-MW DNA-binding protein obtained from: (A), Ada-infected AGMK cells; (Bl, Ad&infected AGMK cells; (0, AdZ-infected KB cells; and CD), Ad5infected KB cells.

in all three of these proteins, indicating that they are likely related proteins. The overall pattern of major P5Slmethioninelabeled peptides from these adenovirus type 12-infected cell DNA-binding proteins (58,000 and 45,000-47,000 MW) bears a resemblance to the adenovirus types 2 and 5 72,000-MW proteins. However the peptide maps of types 2 and 5 72,000-MW proteins appear to be more closely related to each other than either is to the type 12 58,000-MW protein (see Fig. 7). DISCUSSION

The human adenoviruses, types 2,5 and 12, each synthesize single-strand-specific DNA-binding proteins at early times after

productive infection. The molecular weights of these proteins have been determined to be: Ad2, 72,000 and 41,000-45,500; Ad5, 72,000 and 41,000-45,500; Ad12, 58,000 and 45,000-47,000. This paper presents the tryptic peptide analysis of the [35Slmethionine-labeled peptides derived from each of these proteins. Uninfected or mock-infected KB or AGMK cells contain 72,000- and 41,000MW single-stranded DNA-binding proteins that are eluted from a DNA-cellulose column, like the infected-cell proteins, at 1 M NaCI. The peptide maps of the methionine-containing peptides derived from these cellular proteins are distinct from all of the adenovirus type 2, 5 or 12

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FIG. 5. Tryptic fingerprints ot- the mock-mfected, Ad2- and Ad5-induced DNA-binding proteins. Samples were prepared from mock-infected, Ad2- or Ad&infected KB cells as described in the legend to Fig. 1. Regions corresponding to the bands indicated as 72K, A, B, and C in Fig. 1 were cut from the Ad2 gel and the mock-infected control gel, and the eluted peptides processed for fingerprinting as described in Fig. 2. Shown are the autoradiograms corresponding to the fingerprints of: (A), mock-infected KB cell 72,000; (B), mockinfected KB cell 41,000; (C), Ada-infected KB cell 72,000; CD), Ada-infected KB cell 45,000 (Fig. 1, band A); (E) AdP-infected KB cell 41,000 (Fig. 1, band B); (F) AdZ-infected KB cell 37,000 (Fig. 1, band 0.

FIG. 6. Tryptic fingerprints of AdlZ-induced DNA-binding proteins. DNA-binding protein was prepared from Adl2-infected AGMK cells as described, and the 1.0 M NaCl eluant fractionated on SDS-polyacrylamide gels as shown in Fig. 1. Regions corresponding to the three most prominently labeled bands (60K, A’, and B’, Fig. 1) were cut from the gel and processed for fingerprinting as described in Fig. 3. The autoradiograms depict the tryptic fingerprints of (A), the 58,000 AdlZ-induced binding protein, and two smaller AdlB-induced components having approximate molecular weights of(B), 47,000 (band A’), and (0, 45,000 (band B’).

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FIG. 7. An idealized DNA-binding

protein;

tracing of the Ad5, 72,000-MW

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ELECTROPHORESIS major LYSlmethionine-containing DNA-binding protein; Ad12,

proteins obtained from infected cells. This could mean that the adenovirus DNA-binding proteins are indeed coded for by the viral genome. This conclusion is supported by the observation that the adenovirus type 5 72,000- and 45,000-MW proteins are themselves thermolabile for continued binding to DNA when they are obtained from cells infected with H5ts125, a DNAnegative temperature-sensitive viral mutant (van der Vliet et al., 1975). Alternatively, a preexisting cellular protein could be synthesized in larger amounts after adenovirus infection (see Fig. 1). Such a protein might yield a different tryptic peptide map if it were modified in some way. Methylation, phosphorylation, guanylation and adenylation are all possible forms of protein modification that could result in a different peptide map. This alternative appears unlikely because the 72,000-MW adenovirus type 2 DNAbinding protein can be synthesized in vitro from adenovirus-infected cell cytoplasmic messenger RNA (Levine et al., 1975; Anderson, Lewis, Baum, and Gesteland, in preparation). The 41,000-47,000-MW species of adenovirus types 2, 5 and 12 DNA-binding proteins each have peptide maps that are very similar to the larger molecular weight (Ad2 and 5, 72,000; Ad12, 58,000) proteins. The simplest interpretation of these results is that the smaller molecular weight binding proteins are proteolytic breakdown products of the larger proteins. This does not preclude the possibility that these smaller proteins are in fact functional in

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peptides from: Ad2, 58,000-MW DNA-binding

72,000-MW protein.

some manner. An alternative to the proteolysis mechanism could result from an internal start during translation of the mRNA or even an internal promotor (or RNA-processing site) during transcription. The peptide maps of these large and small molecular weight DNA-binding proteins do not permit us to conclude which peptides, if any, might be missing from the small molecular weight proteins. Indeed, since only the methionine-containing peptides are observed P5S-labeled), it is possible that no methionine-labeled peptides are missing when the large and small proteins are compared. The peptide maps obtained here permit one to state only that the patterns are similar enough so that these proteins are clearly related to each other. Finally, these results indicate that the methionine-containing peptides derived from the adenovirus types 2 and 5 72,000MW proteins are more closely related to each other than either is to the 58,000-MW type 12 protein. Based upon the biological, chemical, immunological and genetic evidence (Tooze, 1973) types 2 and 5 human adenovirus appear to be more closely related to each other than to type 12 adenovirus. These data are then consistent with a viral origin of these DNA-binding proteins. ACKNOWLEDGMENTS The

technical assistance of P. Baum, A. Teresky and S. Wyckoff is gratefully acknowledged. We thank R. Gesteland for his advise and critical reading of the manuscript. This research was supported by American Cancer Society Grant No. E591 and in

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part by Public Health Service Research Grant No. CA13106 from the National Cancer Institute of Cold Spring Harbor. REFERENCES ANDERSON, C. W., BAUM, P. R., and GESTELAND, R. F. (1973). Processing of adenovirus 2-induced proteins. J. Viral. 12, 241-252. LEVINE, A. J., KANG, H. S., and BILLHEIMER, F. E. (1970). DNA replication in SV40 infected cells. I. Analysis of replicating SV40 DNA. J. Mol. Biol. 50, 549-568. LEVINE, A. J., VAN DER VLIET, P. C., ROSEN~IRTH, B., ANDERSON, C., RABEK, J., LEVINSON, A., and ANDERSON, S. (1975). Characterizationofanadenovirus early protein required for DNA replication: a single strand specific DNA-binding protein. J. Mol. Cell. Biochem., in press. ROSENWIRTH, B., SHIROKI, K., LEVINE, A. J., and

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SHIM~JO, H. (1975). Isolation and characterization of adenovirus type 12 DNA binding proteins. ViroZogy 67, 14-23. SHIROKI, K., IRISAWA, J., and SHIMOJO, H. (1972). Isolation and a preliminary characterization of temperature-sensitive mutants of adenovirus 12. Virology 49, l-11. TOOZE, J. (Ed.) (1973). “The Molecular Biology of Tumor Viruses.” Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. VAN DER VLJET, P. C., and LEVINE, A. J. (1972). DNA-binding proteins specific for cells infected by adenovirus. Nature New Biol. 246, 170-174. VAN DER VLIET, P. C., LEVINE, A. J., ENSINGER, M. J., and GINSBERG, H. S. (1975). Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Viral. 15,348-354.