Initiation of sequential packaging of bacteriophage P22 DNA

J. Mol.

Biol.

(1982) 157, 287-298

Initiation

of Sequential

Packaging

of Bacteriophage

P22 DNA

SHERWOOD CA~JENS AND WAI MUN HUANG Department

of Cellular,

Viral and Molecular Biology College of Medicine Salt Lake City, Ut. 94132, U.S.A.

University

of Utah

(Received

17 August

1981)

Bacteriophage P22 is thought to package and cut its double-stranded DNA chromosome from concatemeric replicating DNA in a “processive,” sequential fashion. According to this model, during the initial event in such a series the packaging apparatus first recognizes a base sequence, called pat, on the concatemeric phage DNA, and then condenses DNA within the phage head unidirectionally from that point, The DNA is cut at or near the pat site before or during this condensation, and a second cut is made, which separates the packaged DNA from the unpackaged DNA, when the head is full. Subsequent packaging events on that concatemeric substrate molecule then proceed from the end generated by the first event, in the same direction along the concatemer as the first event. In this paper we present evidence that the cuts made at the pat site are in fact not exact, but occur predominantly in six regions, called end sites, located within a 120 base-pair portion of the P22 chromosome.

1. Introduction The packaging of DNA by the double-stranded bacteriophages is currently being studied in considerable detail in several systems. Although the actual physicochemical mechanism by which DNA is condensed within the phage head is not yet fully understood, a substantial body of knowledge concerning the process has been accumulated. Much is known about the numbers, order of action, and overall role of the proteins involved ; but in few cases are molecular details known about the reactions carried out by the individual proteins in this very complex process (Wood & King, 1979; Earnshaw & Casjens, 1980). Phage P22 requires only the products of genes 1,2,3.5 and 8 to accomplish condensation of the phage DNA molecule within the head (Botstein et al., 1973). Of these five proteins, three (gplt, coat protein gp5, and scaffolding protein gp8) are present in precursor particles called proheads, and two (gp2 and gp3) are not found in proheads or in completed phage particles (Botstein et al., 1973; King et al.. 1973; Casjens & King, 1974). In v&o. proheads package chromosomes from concatemeric DNA (head-to-tail tandem repeats of the phage DNA sequence) which arises during replication (Botstein & Levine, 1968: Botstein et al., 1973). The t Abbreviations

used : gpX

refers to the protein

prodwt “87

m2--2836/82/140287-12

$03.00/0

of gene S (gene product

X) : bp. base-pairs.

0 1982 Academic Press Inc. (London) Ltd.

288

S. CASJENS

AND

W. M. HUANG

gene 2 and 3 proteins are thought to be involved in recognition of the proper DNA for packaging, and in nucleolytic cutting which generates the mature length virion chromosome from the overlength replication DNA (see below). gp2 and gp3 purify as a complex (Poteete & Botstein, 1979) and gp2 appears to bind tightly to DNA (Brown & Casjens, unpublished data). Mature P22 virion DNA is a linear double-stranded molecule about 42.000 basepairs in length (Jackson et al., 197%). It is circularly permuted and 30/b (about, 1300 bp) terminally redundant (Rhoades et al., 1968: Jackson et al., 1978u). Tye ef al. (1974), after discovering that the ends of the virion DNA do not actually fall randomly on the genome, but are concentrated within a region of about 15 to 203,. proposed an elegant model for P22 DiYA utilization by the packaging machinery. They enlarged upon the headful packaging model of Streisinger et al. (1967). by suggesting that, on any given concatemeric D1JA molecule. phage DXA is recognized by the presence of a specific DXA sequence (called put by Jackson et cd., 1978a). Recognition is followed by unidirectional condensation from that point. of about 103% of the unit informational sequence into the coat protein shell, and cutting of the headful of condensed DNA from the concatemer. Subsequent events were proposed to proceed in the same direction as the first. from the concatemer end generated by the previous event. Thus, packaging is seen as a series of “processive” events in which several successive non-sequence-specific events follow a sequence-specific one. before the whole process starts over. This model is summarized in Figure 1. The results of Tye et al. (1974). those of
FIG. 1. The sequential packaging model of Tye et al. (1974). Three tandem. head-to-tail repeats of concatemeric phage DNA are shown. The first packaging event (1) begins with recognition of a pae site (a) followed by condensation of DNA in a right-ward direction from that site (it is not known whether this type of an event utilizes pax sites randomly). After a linear headful (about 103% of the sequence between pat sites) of the DNA has been condensed within the coat protein shell it is cut ( 1 ) from the concatemer. The terminal redundancy is magnified about 3-fold in the diagram for clarity. The second event (2) then proceeds right-ward from the end generated by the first event, and the third (3) from the end generated by the second, etc. Thus, if the amount of DNA in a headful is slightly variable (as it appears to be) only the first heedful made in a series will have a specific left end. Notice also that in this model the first event in a series requires 2 double-stranded DNA cuts, while subsequent events require only one.

289

P22 DNA MATURATION

chromosomes (which would happen, if, for example, every packaging event began at a random pat site). There are two types of recognition events in this type of model. (1) The first event in a series which starts with the recognition of a pat site, and (2) subsequent events in which a structure (molecular ends 2) or perhaps proteins left by the previous event are recognized (Tye, 1976). The gene 3 protein is thought to be involved at least

in the first

type,

since mutants

isolated

as having

altered

specificity

for

packaging host DNA (Schmieger & Backhaus, 1976; Chelala & Margolin, 1976) map in gene 3 (Raj et al., 1974) and seem to utilize a new pat site on phage DNA (Jackson

et al., 1982). Furthermore,

only mutants

in genes 2 and 3 block a low level

of in viva DNA cutting in the pat region which occurs in the absence of DXA packaging (Laski & Jackson, 1982), suggesting that these gene products may be directly involved in the cutting of the mature chromosome from the concatemer. This paper concerns the nature of the DNA cuts which occur in the pat region during

normal

DNA

packaging

in viva.

2. Materials and Methods (a) Bacterial and phqr

strain,s

Salmonella typhimurium DB7000 (Winston et al., 1979) was used as the host, and phage particles were purified from cells infected with P22 cl-7 13-amHlO1 (Botstein et al., 1973). (b) Phage LIlVA preparations Phage were purified from infected cells as described (Earnshaw et al., 1976). The phage were dialyzed against 10 m&r-Tris .HCl (pH 7.4). 1 mi!-MgCl, and extracted 4 times against redistilled phenol (Mallinkrodt) which had been equilibrated with 20 m&r-Tris . HCl (pH 78), 1 mM-EDTA. The resulting DNA was dialyzed against 10 m&r-Tris. HCl (pH 78), I mMEDTA, 1 M-NaCl followed by the same buffer without NaCl. (c) Electrophoresis and

recovery

of DLVA fragments

Flat bed agarose electrophoresis slab gels were prepared from a solution of 1.0% (w/v) agarose (SeaKern) in running buffer (40 mivr-Tris-acetate, 5 miw-sodium acetate, 1 mMEDTA. pH 8.0). This mixture was boiled for 3 min and equilibrated to 55°C before pouring. DNA samples were loaded in l/10 concentrated running buffer containing 4 to 9% Ficol400 (Pharmacia). The horizontal gels (13 cm x 20 cm x 95 cm) were run for 270 min at 140 1’. DNA was recovered from these gels by one of two methods: electrophoresing into hydroxylapatite (Tabak & Flavell, 1978) or by the “freeze and squeeze” method of Vogelstein & Gillespie (1979) followed by DEAE-cellulose chromatography. Polyacrylamide gels were run as vertical 13 cm x 16 cm x 915 cm slabs in 91 M-Tris borate (pH 8.3), 1 mM-EDTA. The gels contained 5% (w/v) acrylamide (Eastman), 918% (w/v) bisacrylamide (Eastman) and were polymerized with 0965% (w/v) ammonium per-sulfate and 013% (v/v) N, N, N’N’-tetramethylethylenediamine. Gels were run for 10 min at 150 T followed by 90 min at 250 V. Bands were visualized in both types of gels by staining or, when appropriate, by autoradiography. Gels were stained for 20 min in 025 pg ethidium bromide/ml, destained in deionized water, and photographed with a Polaroid MP-4 land camera through a Wratten 23A filter over a medium wavelength ultraviolet light table (Ultra-Violet Products, Inc.) using Polaroid type 665 positive/negative film. Autoradiography was performed with Kodak X R-2 X-ray film. Quantitation of the intensity of DNA bands on the type 665 negative was

290

8. CASJENS

performed with a Joyce-Loebl3CS graphics calculator.

AND W. M. HUANG

microdensitometer

and a Numonics model 515 electronic

(d) Restriction endonuclease dz$estions Restriction enzymes EcoRI (Miles), ClaI (Boehringer Mannheim), PstI, HpaI, AccI, BstNI (New England Biolabs), HaeIII, HincII, SmaI, XhoI, TaqI, rlva1, HindIII, and BstEII (Bethesda Research Laboratories) were used under the conditions specified by the supplier. Fragment sizes were estimated by comparison of electrophoretic mobility with EcoRIdigested P22 DNA (Chisholm et al., 1980) in agarose gels and TaqI or HaeIII-digested pBR322 DNA (Sutcliffe, 1978) in polyacrylamide gels. (e) End labeling of DNA T4 DNA polymerase was added to label the 3’ ends of double-stranded DNA (Huang & Lehman, 1972). Two units of T4 DNA polymerase (Miles) were incubated with about @5 pg of purified EcoRI put fragment DNA, in the presence of 70 mM-Tris.HCl (pH 8.6), 10 mMMgCl,, 17 micl-ammonium sulfate, 5 mlvr-dithiothreitol, and [32P]dC;TP(@4 Ci/mmol) at 37°C for 9 min. The reaction wa~3stopped by addition of 30 mM-EDTA and heating to 65°C for 3 min.

3. Results Jackson et al. (1978u) showed that, according to the sequential headful packaging model summarized in Figure 1, the cutting of the left end of the initial headful in a series by the packaging apparatus is relatively precise. Upon analysis of restriction fragments in agarose gels, P22 DNA gives rise to a band of DNA which has a restriction cut at one end and a packaging cut at the other. (The right and left ends, respectively, when the DNA is oriented as in Fig. 1.) This fragment, called the “pat fragment” hereafter, is present in submolar amounts because it is generated only in the first of a series of packaging events (Jackson et al., 197%). Every restriction enzyme must give rise to a pat fragment. While analyzing the molar ratio of the pat fragment in phage grown under various conditions (results to be presented elsewhere) it was noticed that the PstI and HpaI pat fragments (both about 2OOO base-pairs long) gave rise to slightly wider bands than neighboring true restriction fragments, suggesting that the packaging cut on the pat fragment might be somewhat imprecise. To pursue this point further, shorter pm fragments were examined, and a restriction map of the region was constructed to give a framework in which to analyze the position of the chromosome ends. The restriction map, shown in Figure 2, was constructed using purified EcoRI pat fragment DNA and the P&I-P22 C fragment (-2210 to 1880 in Fig. 2) or the P&I-P22 F fragment (1880 to 3400) cloned into pBR322 (the last 2 were the kind gift from E. Jackson). Restriction sites were located by analysis of various double digests, partial digests of end-labeled DNA, and digests of various purified sub-fragments of the P&I-P22 C fragment. This was aided by the previous approximate location of the P&I. EcoRI, SWKXI,XhoI, HpaI and Hind111 sites by Jackson et al. (19786), Chisholm et al. (1980) and E. Jackson (personal communication). On the map, the left-most B&E11 fragment was defined as position 400; this allows the chromosome ends to fall near position “0” on the physical map of P22 presented by Chisholm et al. (1980).

P22 DNA

L

“0 I

MATURATION

200

400

600

FIG. 2. Restriction map of the P22 chromosome near the pae region, and location of the chromosome left ends generated during the first packaging event in a series. The P&I, AvaI, SmaI, XhoI, HindIII. ClnI. Hpal, Aeel. EcoRI, B&EII, BstNI, H&II and HaeIII sites in the region were located as described in the text. The scale is in base-pairs. In the construction of the map, the left-most B&E11 site was defined as position 400; this allows the map to coincide with the overall physical map of the P22 chromosome derived by Chisholm et al. (1980). The restriction map is oriented 90 that the initial event in a packaging series begins at about 0 and proceeds right-ward (Jackson et al., 197&z). Below, the map is expanded to show the w fragment sizes produced by HueIII, XhoI and BetEII and the positions. deduced from these sizes, of elzd sites 1 through 6. The height of the vertical arrows on the bottom 3 lines. which designate the various end points of the pat fragments, is approximately proportional to the frequency of cutting at that site (see Table 1 for actual measurements).

Recause they cut P22 DNA only a few times, restriction endonucleases X/WI (left-most cut at position 265), BgtEII (400) and SmaI (465) are useful in this context. since they produce short pat fragments and polyacrylamide gels of the digests are uncongested in the region of the gel where the put fragments migrate. (These enzymes cut P22 DNA 2, 3 and 2 times, respectively; Chisholm et al., 1980; our own unpublished results.) Figure 3 shows P22 DNA digested by these enzymes displayed in a polyacrylamide gel, There is a constellation of at least six bands, the most prominent of which is marked in the Figure, which have the approximate expected (from Fig. 2) mobilities of pat fragments produced by these three enzymes. Notice that the individual bands in these constellations produced by the three enzymes have similar relative intensities. AwnI, which cuts at XhoI and SW& sites as well as others (Roberts, 1980), produces a set of putative pat fragments identical to that produced by X/w1 ; this is consistent with our mapping data which showed no additional AwaI sites between the pat site region and the XbI site. The intense bands which are seen near the top of the gel in the patterns represent the large fragments produced by digestion of P22 DNA by these enzymes. The intense band in the BstEII pattern about 950 bp long results from the BstEII cuts at 400 and 1350. The two small ones in the AvaI pattern result from (1) AwaI cuts at the XhoI (265) and SmaI (465) sites 200 bp apart, and (2) a previously unrecognized AwaI site about 195 bp from one of the seven mapped Am1 sites (E. Jackson, personal communication) between positions of 7000 and 38,000 (there are about, 42,000 bp in the P22 DNA sequence). The faint ladder of bands between 500 and 2000 bp in Figure 3 is of uncertain origin. Although these bands are difficult to see except in grossly overloaded gels, IO

S. CAGJENS

AND u w

P

W. M. HIYANG I

f

-1500

FII:. 3

-

500

-

300

P22 DNA

MATURATION

“!?3

they are reproducibly present in all phage DNA preparations. The details of the patterns are consistent with these being the end fragments which result from “nonsequence-specific” cuts made subsequently to the PC cut in a packaging series, as is the observation that they are not present in the purified EcoRI pat fragment (see below). If this is true, it implies that the position of these subsequent cuts is not truly random or non-sequence-specific; this is under further investigation. If the constellation of bands discussed above is in fact pat fragments, it should be contained within a larger pat fragment. To test this, the EcoRI pat fragment (
FIG. 3. Ijigestion of P22 DNA by restriction enzymes which make short prrc fragments. The 5 central lanes contain phage P22 DNA digested with the enzymes indicated. The left lane contains pBR322 HneIII and TnqI fragments for molecular weight markers, and the right lane contains pBR322 HneIII fragments. To the right of the gel is a scale derived from these standards which relates mobility to fragment size in base-pairs. Electrophoresis of DNA fragments in 5% polyacrylamide is described in Materials and Methods. The white arrowheads mark the most intense band of the set of bands thought to br in the pnc fragment group. and the vertical white lines adjacent to the AvaI. B&II and Smol lanes denote the extent of the constellation of pnc fragments in these lanes.

LSJENS BsfEU

AND +

W. M. HI’ANG

XhoI

.

I

EcoRI

pm

P22 fragment Flc:

4

pBR322

P22 DNA

MATI’RATIOS

TABLE 1 Frequency

md cutting region 1 2 3 4 5 6

and position

of cuts in the pat regiort

Relative cutting frequencyt Phage DNA EeoRI pnc fragment oa + 0.02 0.16?@02 0.36 k 004 0.11* 002 0.2 1 * 0.03 oa6 * 602

04M &-0.02 @17*@03 032 * 003 614iOO2 623 k 094 oa9 * 002

Map position1 -6Of3 -22+3 1 *3 20*3 38f3 55+3

t Determined by measuring the relative intensity of the pat fragment bands produced by packaging cuts at the different cutting sites and correcting for their molecular weights. P22 phage Dn’A and purified EcoRI put fragment DNA were used. In each case the uncertainties represent the span of values obtained in at least 6 determinations, including 2 each using B&II. SmnI and XhoI cut DNA. $ Determined (using HaeIII. AvaI and XhoI) by subtracting the length of the pnc fragments from the map position of the restriction site to the right which. when cut. generates the fragment (Fig. 2). The uncertainties show the approximate range of values obtained in several determinations with each enzyme. HincII. RslNI. SmaI and B&E11 values agree with these positions. but are less accurate because these pat fragments are considerably larger.

of course limited by whether or not the pat fragments run true to their sizes in polyacrylamide gels. The relative frequencies of cutting at these sites were determined by measuring the relative molar intensities of the various pnc fragments (data not shown). The results of these calculations are given in Table 1. It is quite possible that each end cut is itself imprecise: some of the XhoI and HneIII pat fragment bands appear more diffuse than nearby true restriction fragments (i.e. cuts at individual end “sites” or regions might be spread over up to 3 to 5 bp. particularly at end 2 and end 6). Are all of the “pat cuts” made within this 120 bp region? Several arguments suggest that most are. First, autoradiograms from long exposure of gels of endlabeled pat fragments (e.g. see Fig. 4) do not show fragments outside this region. Second, quantitative determinations of the sum of the ethidium staining material between 200 and 325 bp and that in the 135 bp XhoI-BstEIT band in a double digest (as in lane 4. Fig. 4) of pure EcoRI pm fragment gave a pat fragment/l35 bp fragment molar ratio of l-04 + 0.08. Thus, the number of molecules in the XhoI pnc fragment envelope is very close to the number of molecules of E’coRI pat fragment which were digested. When the EcoRI pat fragment for this analysis was isolated from gels by the method of Vogelstein & Gillespie (1979), a fairly large region (including the major pat fragment) was cut from the gel to include possible minor

Frc:. 4. Restriction enzyme analysis of end-labeled EcoRI yc fragment. The puritied KcoRI pcrc fragment was end-labeled with [a-32P]dCTP as described in Materials and Methods and digested with R&E11 or a combination of B&E11 and XhoI as indicated in the Figure. The left portion of each of the left 2 pairs of lanes is an autoradiogram of the resulting 5% polyacrylamide gel. and the right is a parallel lane stained with ethidium bromide. On the right are mature P22 DNA cut by XhoI and pBR322 Hoe111 fragments as size markers and a scale in base-pairs derived from them.

296

6. CASJENS

AND

W. M. HL’AXG

pat fragments not migrating exactly coincident with the visible EcoRl pat fragment band. Therefore, a significant fraction of XhoI pat fragments is not being lost in the background of the gel by being too spread out to visualize. or bl- being left behind during the original pat fragment isolation. Finally. Aval digests such as the one shown in Figure 3, lane 2, allow the quantitation of the fraction of the total P22 DNA molecules which give rise to fragments that migrate in the pat fragment constellation. The ratio of the molar intensities of the 200 or 195 bp fragment to the pat fragment constellat,ion in :l~‘oI digested P22 DNA was 3.5 kO.7 in three determinations (gel scans not shown). If the assumption is made that the 200 or 195 bp fragments are rarely cut by the packaging machinery. then nearly every DNA molecule gives rise to each of these fragments. while only the first DNA molecule in each packaging series gives rise t’o a pat fragment (Jackson et al.. 19786). The measured ratio implies an average series length of about 3.5, which is in good agreement with previous series length determinations (Tye et aI., 1974; Jackson et nl., 1978u). Thus. the =IvnI p~c fragments are present in the expected amounts.

4. Discussion The experimental results presented here suggest that the phage P22 DKA packaging apparatus does not cut DNA at a unique position when it initiates a sequential series of packaging events: rather, cuts are scattered within a 120 bp region. Furthermore, these “scattered” cuts are not random, but cluster around the six sites on the chromosome called end 1.2, 3,4,5 and 6 (see Fig. 2 and Table 1). It’ is curious that they are separated by intervals of about 20 bp (except 1 and 2 which are separated by an approximate multiple of 20 bp). Thus. according to the sequential packaging model (Tye et al.. 1974) each time the P22 DNA packaging apparatus initiates a sequential series of packaging events. it has a choice of cutting in one of these end regions. It is not yet entirely clear whether or not actual condensation of DNA within a prohead is required for these cuts to occur in V~UO. Laski & Jackson (1982) have presented data consistent with the idea that at least some cuts in the pat region can occur in the absence of proheads and therefore in the absence of DNA packaging. They have further shown that these DNA packaging-independent cuts are entirely dependent upon the phage gene 2 and gene 3 products. It is not yet known whether these cuts which occur in ~:ivo without DNA packaging are made at the end sites defined in this paper. A possible alternate model. that, “pat ends” represent concatemer ends resulting from replication has been made unlikely by the results of Weaver & Levine (1978) and Laski & Jackson (1982). H. Schmeiger & H. Backhaus (personal communication) have recently sequenced the DNA in this region and found no clear repeating sequence which correlates with the positions of the end sites. It seems likely. since P22 packages replicated P22 DNA more efficiently than host DNA (Ebel-Tsipis et al., 1972) and since t,he packaging reaction occurs unidirectionally along the Dh’A (Jackson et al., 197% : Weaver & Levine, 1978), that there must be a specific nucleotide sequence in the phage chromosome which the phage packaging apparatus recognizes as a signal t,o

P22 DNA

MATURATION

297

initiate DNA condensation within the prohead. We propose to reserve the name “r)ac site” for this recognition site, whose detailed location remains unknown but which is likely to be within a few kilobases of the end sites (Weaver & Levine, 1978 : B. Kufer, H. Backhaus & H. Schmieger, personal communication). How could the recognition site be different from the cutting sites? Two models seem most plausible : (1) the packaging apparatus can move by approximate multiples of 20 bp (2 helix t,urns ?) after recognition, but before cutting; or (2) after the packaging apparatus has recognized and bound the pat site. additional protein molecules (gp2 and/or gp3 7) bind adjacently along the DNA in a non-sequence-specific fashion, and any of these bound proteins may be the one which cuts the DNA. It is interesting to note the parallels between this and the phage lambda packaging apparatus which always makes a single, unique cut in the DNA at a site called cos (Wu & Taylor, 1971: Becker & Gold. 1978). The lambda terminase (that protein which actually creates the molecular termini found in mature phage chromosomes: Mousset & Thomas, 1969: Earnshaw & Casjens, 1980) appears to consist of a complex of two polypeptides, the products of the Nul and A genes, which have molecular weights of 21,500 and 79,000. respectively (Sumner-Smith et /I/.. 1981). The putative P22 terminase (Laski & Jackson, 1982; Casjens & Brown. unpublished data) is also a complex of two proteins, the gene 3 and gene 2 proteins (Poteet’e 61 Rotstein, 1979), which have molecular weights of 17,000 and 67,000. respectively (Youderian & Susskind, 1980). Jackson et al. (1978a) previously noted that the P22 gene 2 and gene 3 occupy positions on the genetic map which are analogous to the gene A and gene Nul of lambda. Feiss et al. (1979) have shown genetically that, in addition to the 22 bp region with rotational symmetry which is cut by t’hr lambda terminase (Weigel et al.. 1973), a DNA sequence which is nearby and to the right of the cutting site is required for in viuo recognition of the DNA as a suitable packaging substrate. This is somewhat reminiscent of the model we have suggested for P22 recognition, in that the cutting and recognition sites may not be congruent. Furthermore, Feiss et al. (1979) also found that the presence of this sequence is not required at the cos site at the right end of the molecule being packaged, suggesting that a cos site at a packaging recognition-initiation event is handled by the packaging apparatus differently from the cos site at the other end of the LISA molecule. Laski & Jackson (1982) have suggested that the cutting at the right and left ends of P22 during packaging is also not identical ; however, it should be stressed that they found that lambda never cuts at cos unless DNA is being packaged, whereas P22 sometimes cuts near pat in the absence of DNA packaging. Finally. Emmons (1974) and Feiss & Bublitz (1975) showed that lambda, like P22, packages DNA in a sequential. “processive” manner from concatemeric DNA in r+c:o. Thus. it seems clear that. in spite of the obvious differences in sequence specificity of cutting, the P22 and lambda terminases have a number of basic similarities. We thank E. Jackson, H. Schmieger and A. Becker for communicating their unpublished results to us, E. Jackson for kindly providing strains carrying plasmids containing cloned I’22 restriction fragments and D. Carroll for providing several restriction enzymes. This work was supported by National Institutes of Health grant GM2195, National Science Foundation grant PCM78-1373, and a National Institutes of Health Career Development, Award (to S.C.) and National Institutes of Health grant GM21960 (to W.M.H.).

298

6. (‘ASJENS

AND W. M. HVANG

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by A. Kluq