Newcastle disease virus RNA

J. Mol. Riol. (1966) 18, 204-214

Newcastle Disease Virus RNA ll. Preferential Synthesis of RNA complementary to Parental Viral RNA by Chick Embryo Cells DAVID

W.

KINGSBURY

Laboratory of Virology, St. Jude Children's Research Hospital and University oJ Tennessee, Memphis, Tennessee, U.s.A. (Received 10 November 1965, and in revised form 24 February 1966) Following infection by strain "0" of Newcastle disease virus, and during incubation in the presence of actinomycin D, chick embryo cells synthesized RNA which had a base composition complementary to the base composition of RNA from virus particles. Evidence for base-sequence complementarity between RNA from virus particles and RNA synthesized by infected cells was obtained by in vitro hybridization. Of the total radioisotopically labeled RNA synthesized by infected cells in the presence of actinomycin D, 80% or more became insensitive to pancreatic ribonuclease after annealing with RNA from virus particles. The ribonuclease-resistant RNA had properties of a double-stranded polynucleotide. RNA from chick embryo cells or from Escherichia coli cells did not render labeled RNA from Newcastle disease virus-infected cells resistant to ribonuclease, and RNA from virus particles did not confer ribonuclease resistance on RNA from uninfected chick embryo cells. From 18 to 40% of the labeled RNA obtained from infected cells became RNase-resistant when annealed in the absence of RNA from virus particles. Cells infected by any of four other strains of Newcastle disease virus synthesized RNA, which was complementary in base composition to RNA from strain "0" virus particles. Virus particle RNA from all of these strains hybridized with RNA from cells infected by the "0" strain.

1. Introduction Cells infected by NDVt synthesize RNA in the presence of concentrations of actinomycin D which inhibit cellular DNA-dependent RNA synthesis more than 95% (Kingsbury, 1962; Wheelock, 1963). This virus-induced RNA synthesis parallels virus assembly, but there are indications that less than 10% of the RNA synthesized by infected cells appears in progeny virus (Kingsbury, 1962; Wheelock, 1963). The base composition of the RNA synthesized by infected cells in the presence of actinomycin D was reported to be similar to the base composition of the RNA in NDV particles (Scholtissek & Rott, 1964). This implies that infected cells synthesize predominantly RNA that resembles parental viral RNA, as shown for some other H'NA viruses (Darnell, 1962; Scholtissek, Rott, Hausen, Hausen & Schafer, 1962).

t Abbreviations used: NDV, Newcastle disease virus; SDS, sodium dodecyl sulfate; 0·15 M-NaCI, 0·015 M-sodium citrate. 204

sse,

NEWCASTLE DISEASE VIRUS A COMPLEMENTARY RNA

205

However, as will be shown, the base composition of the RNA synthesized in the presence of actinomycin D by cells infected by the "0" strain of NDV is complementary to the base composition of the rapidly sedimenting RNA isolated from virus particles. Further, it is shown that most of the RNA synthesized by infected cells hybridizes in vitro with RNA from virus particles, indicating complementarity of nucleotide sequences.

2. Materials and Methods (a) Virus Procedures for production and purification of the "C" strain of NDV have been described (Kingsbury, 1966). (b) Determination of base composition of RNA from infected cells

Primary chick embryo cell monolayers were prepared as described by Granoff (1959). After 2 days of growth at 37°C, monolayers were inoculated with 20 plaque-forming units of virus per cell. After 30 min at 37°C, the inocula were removed and replaced with bicarbonate-buffered, phosphate-free basal salt solution (Hanks, 1948) containing 0'5% (wjv) lactalbumin hydrolysate, 100 to 200 fLcjml. carrier-free [32P]0~ -, and 10 flg/ml. actinomycin D. After 8 hr further incubation at 37°C in an atmosphere of 5% CO 2 in air, the medium was removed and the cells were disrupted by adding 0'5% (wjv) SDS in 0·01 M-sodium acetate, 0·05 M·NaCI (pH 5,1). Suspensions of disrupted cells were transferred to test tubes and shaken for 5 min with equal volumes of water-saturated phenol at 60°C (Scherrer & Darnell, 1962). After centrifugation at 5000 g for 10 min at 2°C, the aqueous layers were removed and shaken with fresh phenol. RNA precipitated from the final aqueous phases at - 20°C after addition of 2 vol, of ethanol and 0·2 vol, of 5 M·NaCl. RNA was separated from low molecular weight contaminants by passage through 1 cm X 20 em columns of Sephadex G25 equilibrated with 0'01 M·sodium acetate, 0·05 M· NaCI (pH 5'1). Two column passages were usually necessary. RNA was concentrated by ethanol precipitation and hydrolyzed in 0·3 N·KOH at 37°C for 18 hr. After hydrolysis, solutions were neutralized with perchloric acid. Ribonucleotides were separated by electrophoresis on Whatman no. 3 MM paper strips in 0·01 M-sodium citrate (pH 3,5) at 67 vlcix: in a fiat-plate system for 90 min. The electropherograms were dried and cut into 1-cm segments. Segments were placed in vials containing a toluene.based scintillation phosphor and counted in a liquid-scint.illation spectrometer. Four zones of radioactivity were always obtained, corresponding in mobility to marker RNA ribonucleotides. (c) Preparation of tritium·labeled N D V -induced RNA Infection of chick embryo monolayers was performed as described above. Virus inocula were replaced by bicarbonate-buffered Hanks basal salt solution containing 0'5% (w/v) lactalbumin hydrolysate, 10 flg/m!. actinomycin D, and 10 fLc/m!. [3H]uridine (specific activity: 5 ejm-mole). After 6 to 8 hr at 37°C in an atmosphere of 5% CO 2 in air, cells were disrupted with 0'5% (wjv) SDS in 0·01 M-sodium acetate, 0·05 M-NaCI (pH 5,1), and RNA was extracted with phenol at 60°C. One passage of an RNA solution through a 1 cm X 20 em column of Sephadex G25, equilibrated with 0·1 M-ammonium acetate (pH 5'5) separated it from all acid-soluble radioactive material. Column fractions containing RNA were pooled and stored at - 20°0. (d) Preparation of rapidly sedimenting RNA from N D V particles

Virus particle RNA was isolated as previously described (Kingsbury, 1966), using an agarose column equilibrated with 0·1 M.ammonium acetate (pH 5'5).

(e) Preparation of E. coli ribosomal RNA and chick embryo cell RNA Escherichia coli ribosomes were isolated by the method of Nirenberg (1963). Chick embryo cells were grown as monolayers for 2 days at 37°C. RNA was extracted by the 14

D. W. KINGSBURY

206

hot-phenol method (Scherrer & Darnell, 1962), and purified by passage through G25 Sephadex columns equilibrated with 0·1 M-ammonium acetate (pH 5'5). (f) Procedure for annealing RNA and for determination of

RNase-resistant radioactive material RNA samples in 0·1 M-ammonium acetate (pH 5'5) were lyophilized in 0·5 em X 10 em tubes of soda-lime glass. The RNA was dissolved in 0·025 ml. of 2 X sse per tube and the tubes were sealed by flame. Samples were heated in an oil bath at 800e for 1 hr, then cooled to 23°e over a 5-hr period. To determine RNase-resistant radioactive material, each sample was diluted to 1·0 ml. with 1 X sse, and a 0·5-ml. portion was treated with 1 to 10 f'gfml. RNase A (Worthington Biochemicals, lot RAF 6079) for 30 min at 23°e. The remaining 0·5 ml. was held at 23°e as an RNase-free control. Following enzyme treatment, 3 ml. of 5% trichloroacetic acid and 200 f'g bovine serum albumin were added to each sample. After 30 min at 4°e, precipitates were collected on type HA Millipore filters and washed with acid. The filters were dried, placed in vials containing a toluene-based scintillation phosphor, and counted in a liquid-scintillation spectrometer.

3. Results (a) Base composition of RNA from N D V particles compared with the

base composition of RNA from infected cells

The base composition of the rapidly sedimenting RNA from strain "0" NDV particles and the base composition of the RNA synthesized by infected cells in the presence of 10 p.gjml. actinomycin D are not similar, but they appear complementary (Table 1). TABLE

1

RNA base compositions Mole per cent Source of RNA C

A

G

U

NDV particles strain "C"

23·2 (± 0,2)

20·1 (± 0,6)

25·4 (± 0,6)

31·2 (± 0,6)

Cells infected by strain "C"

25·8 (± 0,6)

30·4 (± 0,8)

22·3 (± 0,4)

21·5 (± 0·4)

Calculated complement of strain "C" particles

25·4 (± 0,6)

31·2 (± 0,6)

23·2 (± 0,2)

20·1 (± 0,6)

The base composition of RNA from NDV particles compared with the base composition of 32p. labeled RNA from infected cells incubated in 10 fLgfml. actinomycin D. The figures in parentheses represent standard deviations of the means of 4 or more determinations. The calculation of base composition complementary to the RNA of NDV particles was based on the relations: A = U. G=C.

(b) Annealing test of complementarity

To determine whether the statistical evidence of base complementarity between RNA from virus particles and RNA from infected cells reflected complementarity of nucleotide sequences, these RNA's were mixed and annealed. Annealing made most of the labeled RNA from cells resistant to RNase (Table 2, column 3). Labeled RNA in mixtures which were not annealed was sensitive to RNase (Table 2, column 2), showing that the unlabeled viral RNA did not inhibit the enzyme non-specifically.

NEWCASTLE DISEASE VIRUS A COMPLEMENTARY RNA TABLE

207

2

Annealing of 3 H-labeled N D V-induced ENA with ENA from N D V particles RNase-resistant radioactive material (% of control)

RNA species

Mixed at 23°C

Annealed

180 p.g/m!. ["H]RNA from infected cells + 40 ""g/m!. NDV particle RNA

5·3

93·2

180 p.g/mI. [3H]RNA from infected cells alone

6·3

34·8

Samples of RNA from infected cells were mixed with RNA from NDV particles and either incubated in 1 ml. I X SSC at 23°C for 30 min or annealed in 0·025 ml. 2 X SSC at 80 0 0 . Portions of each reaction mixture were treated with RNase, and RNase-resistant radioactive material (cts) was compared with controls not treated with RNase before precipitation with acid.

(c) ENase resistance produced by -annealing ENA from infected cells alone

When RNA from infected cells was annealed without RNA from virus particles, some RNase resistance was acquired (Table 2). From 18 to 40% of the labeled RNA became RNase-resistant in different preparations, but the amount of RNase resistance obtained with each preparation in separate trials was constant. Varying the amount oflabeled RNA did not change the proportion oflabel rendered RNase-resistant by annealing (Table 3). The unlabeled cellular RNA which was present in labeled RNA preparations was apparently not involved in this reaction, since additional quantities had no effect (Table 4). TABLE

3

Effect of ENA concentration on ribonuclease resistance of the product of annealing ENA from infected cells ""gJm!. RNA

RNase-resistant radioactive material (% of control)

40 400 4000

22·0 18·0 25·8

After lyophilization, 3H-labeled RNA from infected cells was dissolved in 2 X SSO and annealed. Each sample was then diluted in 1 X SSC to give a final concentration of 4 ""g/ml. RNA and treated with RNase. Acid-precipitable radioactive material after enzyme treatment was compared with acid-precipitable radioactive material in RNase-free controls.

(d) Specificity of the reaction of viral ENA with ENA from infected cells

Low concentrations of virus particle RNA conferred ribonuclease resistance above the background level on RNA from infected cells, whereas 1000-fold greater concentrations of E. coli ribosomal RNA did not (Fig. 1). RNA from virus did not react with labeled RNA from uninfected cells (Table 5).

D. W. KINGSBURY

208

4

TABLE

Effect of added RNA from chick embryo cells on the RNase-resistant product of ann ealing in the absence of RNA from virus particles RNase resistant radioac t ive material (% of con trol)

RNA Species

400 p.gJml. [3H]RNA from infected cells 1600 p.g /ml. ch ick cell R N A

+ 38·6 40 ·7

400 p.g/ml. [3H]RNA from in fec te d cells

After annealing, portions were treated with RNase, and ac id -p re cipitable radioactive m a t erial (cts) was compared with acid-precipitable material from controls n ot treated with enzyme.

2

100 10

/ ,

10°

I I IIII

80

- -e--- ----.--~- _ < ,

101

102

103

Unlabeled RNA added "'9 / mi.l

FIG . 1. Annealing of NDV-in duced RNA w it h RNA from NDV particles or E . coli ribosom al RNA. Samples of 3H -labe led NDV·induced RNA (400 p.g/ml.) we re anneal ed with purified high molecular weight RNA fro m viru s particles or E . coli ribosom al RNA at the indicated concentrat ions. A cid-precipitable radioactive material of each sam p le after RNase d igestion was compared with a cid-precipitable radioactive m aterial of an undigested con t r ol. Annealed with RNA from virus particles; annealed with E. coli ribosomal RNA.

--e-e-,

--e--e--,

TABLE

5

A nnealing of RNA fr om virus particles and RNA from uninfected cells RNase-resistant ra d ioa ct iv e material (% of contro l)

RNA sp ecies

320 p.g/ml. [3H]RNA from un in fec te d cells 40 p.g /mI. NDV particle RNA 320 p.g /ml. [3H]RNA fr om uninfect ed cells

+ 10·2 13·4

3H -labele d cell RNA w as prep ared fr om uninfected cells in cu bated for 6 hr in 10 p.c /ml. [3H ]_ uridine. After annealing, porti ons we re t reate d with RNas e , a n d ac id -p recipi t ab le radioactive material (cts) was compared with con tr ols not treated with RNase.

NEWCASTLE DISEASE VIRUS A COMPLEMENTARY RNA TABLE

209

6

Effect of DNase pretreatment on ribonuclease-resistant products of annealing

RNA Species

RNase resistant radioactive material (% of control)

400 p.gjrnl. [3H]RNA from infected cells + 200 p.g{ml. NDV particle RNA

94-5

400 p.g{ml. [3HJRNA from infected cells

35·9

Labeled RNA from infected cells was incubated with 50 p.g{ml. pancreatic DNase in 0-001 ~l­ Mg 2 + for 30 min at 23°C before lyophilization and annealing. After annealing, acid-precipitable radioactive material of samples treated with RNase was compared with acid-precipitable radioactive material of controls which did not receive enzyme.

Contribution of DNA to the RNase-resistant label or to its production by annealing was ruled out by pretreatment of labeled RNA from cells with DNase (Table 6). (e) Properties of RNase-resistant products of annealing

The RNase-resistant material produced by annealing labeled RNA from infected cells with optimal amounts of virus particle RNA had a sharp thermal transition to RNase sensitivity between 95°C and HO°C (Fig. 2(a», indicating a double-stranded structure (Geiduschek, Moohr & Weiss, 1962; Gomatos & Tamm, 1963). In contrast, the RNase.resistant material produced by annealing RNA from cells alone lost 30% of its resistance to the enzyme between 60°C and 97°C (Fig. 2(b». The RNase sensitivity of the cellular RNA annealed by itself was more dependent on enzyme concentration than the annealed mixture at RNase concentrations less than 10 p.gfml. (Fig. 3). RNase resistance of the specifically annealed RNA mixture was dependent on salt concentration (Fig. 4), as expected for a double-stranded polyribonucleotide (Warner, Samuels, Abbott & Krakow, 1963). (f) Oomplementary RNA in four other N D V strains

The base compositions of RNA's synthesized in the presence of actinomycin D by cells infected with any offour other strains ofNDV are similar to the base composition of RNA synthesized by cells infected with strain "C" (Table 7). In the presence of 10 fLgfml. actinomycin D, uninfected cells incorporated little labeled precursor into RNA, recovery of label chiefly in cytidylic and adenylic acids suggesting that incorporation was primarily into the acceptor terminal nucleotides of amino acid transfer RNA (Franklin, 1963). When RNA from virus particles of each of these NDV strains was annealed with labeled RNA from cells infected by the "0" strain, conversion to RNase resistance occurred (Table 8), indicating that RNA's from these strains of NDV are similar to RNA from "0" strain virus in nucleotide sequences.

100

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80

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60

15

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1c: -0

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100

50

130

(b)

FIG. 2. (a) Thermal denaturation of the product of annealing RNA from NDV particles with RNA from infected cells. [3HJRNA from infected cells (180 fLgjml.) was annealed with RNA from virus particles (40 fLg/ml.). Mter annealing, the mixture was diluted 200-fald in 1 x sse, and portions were sealed in glaas tubes. Samples were incubated at the indicated temperatures for 10 min, cooled rapidly to - 3°C, and treated with RNase. Heated and cooled samples which did not receive RNase served as controls. (b) Thermal denaturation of the product of annealing RNA from infected cells without virus particle RNA. RNA from infected cells was annealed, diluted in 1 x sse, heated and cooled as described in the caption to (a). Acid-precipitable radioactive material of RNase-treated samples was compared with acid-precipitable radioactive material of controls which did not receive RNase.

100 _.

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~o

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80

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60

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10 R: 60 nuclease {fig I ml.l FIG. 3

100

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0·10 /' 0'30 0·05 Salt concentration (M-NaCl; 10'1 M-sodium citrate) FIG. 4

FIG. 3. Effect of RNase concentration on acid-precipitable radioactive material in samples of [3H]NDV-induced RNA annealed with RNA from virus particles and annealed without added RNA. The concentration of [3H]NDV-induced RNA in each annealing reaction was 108l'g/ml. Annealed samples were diluted 40-fold in 1 X sse and incubated with RNase at 23°e for 30 min. Annealed with 40 I'g/rnl. virus particle RNA; --e--e--. annealed without virus particle RNA.

-e-e-.

FIG. 4. RNase resistance of the RNA hybrid at different salt concentrations. Samples of annealed RNA obtained as described in the caption of Fig. 2(a) were diluted 200-fold in sse at the indicated salt concentrations. and incubated with RNase. Enzyme-free samples were the controls.

212

D. W. KINGSBURY

7

TABLE

RNA base compositions Mole per cent

Strain of NDV

DelawareHickman

26·5

N.J.-Roakin

27·8

(± 0,6) (± 0,3)

KansasLeavenworth

26·5

Texas-GB

27·6

(± 1,2) (± 0·4)

Uninfected cells

A

C

48·7 (± 1-5)

G

34·5

U

19·8

(± 0'5)

19·2

(± 0'5)

29·9

21·1

(± 0,9)

20·8

(± 1,3)

20·9

(± 0,8)

5·5

33,000

(± 0,8)

41,400

(± 0,6)

42,300

8·3

(± 0,7)

(± 1'7)

(± 0,1)

20·6

(± 0,5)

37·4

26,400

21·4

(± 0,4)

30·8

(± 0,6)

21·3

(± 0,3)

31·3

Total radioactivity (ctsJmin)

(± 0,6)

1660

Base compositions of 32P-labeled RNA's synthesized by chick embryo cells in the presence of 10 p.gJml. actinomycin D after infection by each of several strains of NDV. Figures in parentheses represent standard deviations of the means of 5 or more determinations.

TABLE

8

RNase resistance produced by annealing RNA from virus particles of other N D V strains with RNA from cells infected by the' '0" strain RNase-resistant radioactive material (% of control)

RNA species

+

120 p.gjml. [3H] RNA from "C" infected cells 80 p.g{ml. Kansas virus particle RNA

96·6

+

120 p.gjml. [3H] RNA from "C" infected cells 80p.gjml. Hickman virus particle RNA

97·2

+

120 p.gjml. [3H] RNA from "C" infected cells 80 p.gJml. Texas virus particle RNA

93·8

120 p.g{ml. [3H] RNA from "C" infected cells + 80 p.gjml. N.J. virus particle RNA

94·9

120 p.g{mI.[3H] RNA from "O" infected cells

21·3

Rapidly sedimenting RNA was obtained from particles of each of the NDV strains as previously described (Kingsbury, 1966). After annealing, samples from each reaction mixture were treated with RNase. Acid-precipitable radioactive material was compared with acid-precipitable radioactive material in RNase-free controls.

NEWCASTLE DISEASE VIRUS A COMPLEMENTARY RNA

213

4. Discussion The data indicate that NDV caused cells to synthesize RNA complementary in base sequence to the RNA in virus particles. In the presence of actinomycin D, production of the complementary RNA exceeded production of parental RNA replicas. Although parental RNA was not measured in cell extracts, it is reasonable to assume that some was present, since chick embryo cells produce virus in the presence of actinomycin D (Kingsbury, 1962). Preferential synthesis of complementary RNA may explain why only a fraction of the RNA synthesized by infected cells in the presence of actinomycin D was recovered in virus particles (Kingsbury, 1962; Wheelock, 1963). While the base compositions and the annealing results show that complementary RNA is predominant in cell extracts, the precise percentage cannot be stated. This awaits determination of how much, if any, complementary RNA is in the fraction made RNase-resistant when RNA from cells is annealed by itself. This RNase-resistant product of annealing without unlabeled RNA from virus particles might result from hybridization of labeled parental RNA molecules with some complementary molecules. Yet this could explain only part of the RNaseresistant material, since some was digested by concentrations of enzyme which had no effect on the specifically hybridized RNA (Fig. 3), and some became RNasesensitive at lower temperatures than the specific hybrid (Fig. 2). Another possibility is that the complementary RNA can hybridize with itself. Such a reaction might be limited to specific nucleotide sequences, since the percentage of RNase-resistant material was not dependent on the concentration of RNA (Table 3). Scholtissek & Rott (1964) reported that the base composition of RNA from NDV "Italien" was similar to the base composition of RNA from infected cells. These base compositions are like the base compositions of RNA's obtained from cells infected with any of the five strains of NDV tested in the present work. Oontamination of virus with RNA from cells might explain the results of these workers, or the "Italien" strain of NDV may behave differently. If the complementary RNA is not an artifact produced by actinomycin D, what is its function in replication of the virus? It could be a template for replication of parental viral RNA, as suggested for the complementary RNA's found in double-stranded "replicating forms" of other RNA viruses (Ochoa, Weissman, Borst, Burdon & Billeter, 1964). However, since the complementary RNA synthesized in NDV infection is abundant and single-stranded, a different function is suggested. This complementary RNA might be a template for viral protein. This work was supported by U.S. Public Health Service research grant AI-05343 from the National Institute of Allergy and Infectious Diseases and by the American Lebanese Syrian Associated Charities (ALSAC). I am a career development awardee of the U.S. Public Health Service (5K3 HD-14,(91). The guidance of Dr A. Granoff is gratefully acknowledged. Mr .Tames East provided skilled technical assistance. REFERENCES Darnell, .T. E. (1962). Cold Spr, Barb. Symp. Quant. BioI. 27, 149. Franklin, R. M. (1963). Biochim. biophys. Acta, 72, 555. Geiduschek, E. P., Moohr, .T. W. & Weiss, S. B. (1962). Proc, Nat. Acad. Sci., Wash. 48, 1078. Gomatos, P. J. & Tamm, 1. (1963). Froc. Nat. Acad. Sci., Wash. 49, 707.

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w,

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Granoff, A. (1959). Virology, 9, 636. Hanks, J. H. (1948). J. Oell. Oomp. Physiol. 31, 235. Kingsbury, D. W. (1962). Biochem, Biophys. Res. Oomm. 9, 156. Kingsbury, D. W. (1966). J. Mol. Biol. 18, 195. Nirenberg, M. W. (1963). In Methods in Enzymology, ed. by S. P. Colowick & N. O. Kaplan, vol. 6, p. 17. New York: Academic Press. Ochoa, S., Weissman, C., Borst, P., Burdon, R. H. & Billeter, M. A. (1964). Fed. Proc, 23, 1285. Scherrer, K. & Darnell, J. E. (1962). Biochem, Biophys. Res. Comm, 7, 486. Scholtissek, C. & Rott, R. (1964). Virology, 22, 169. Scholtissek, Co, Rott, R., Hausen, Po, Hausen, H. & Schafer, W. (1962). Oold Spr, Harb. Symp. Quant. Biol. 27,245. Warner, R. Co, Samuels, H. H., Abbott, M. T. & Krakow, J. So (1963). Proc, Nat. Acad. Soe., Wash. 49,533. Wheelock, E. F. (1963). Proc. Soc. Exp. BioI. Med. 114, 56.