The effect of extraction protocol on the yield, purity, and translation products of RNA from an isometric plant virus

Journal

of Virological

Methods,

10 (1985)

195

195-202

Elsevier JVM 00373

THE EFFECT OF EXTRACTION PROTOCOL ON THE YIELD, PURITY, AND TRANSLATION PRODUCTS OF RNA FROM AN ISOMETRIC PLANT VIRUS

M.J. BRISCO*.

R. HULL

John Innes Institute.

(Accepted

Colney

29 October

This is a comparative for translation shaking chloride;

preparing

Lane,

dissociated

U.K.

19X4)

study of methods

used to extract

were: removal

of RNA from protein

of different

to different

methods

by sucrose

yields and purities.

comparison

bean mosaic virus (SBMV)

either by 4 M sodium precipitation

density gradient

On translation

extents, and gave different

SBMV RNA for in vitro translation

RNA preparation

RNA from southern

of protein,

virus with a mixture of phenol and chloroform;

or separation synthesis

WILSON

Norwich,

in vitro. The methods

gave RNA samples protein

and T.M.A.

perchlorate

centrifugation.

These methods

in vitro the RNA samples

sets of polypeptide

products.

in vitro translation

stimulated

The best method for

was that which used the phenol-chloroform

of methods

or by

of RNA from 2 M lithium

southern

mixture.

beanmosaicvirus

INTRODUCTION

Southern bean mosaic virus (SBMV) is an economically important pathogen of legumes. It has isometric particles of relative molecular mass 6.6 X lo6 (Miller and Price, 1946) and icosahedral T = 3 geometry (Johnson et al., 1976). The genome consists of one species of positive-sense, single-stranded RNA of relative molecular mass 1.4 X lo6 (4,500 nucleotides) although smaller molecules are also encapsidated (Mang et al., 1982). The in vitro translation of SB?/lV RNA has been studied by Rutgers et al. (1980), Salerno-Rife et al. (1980), Ghosh et al. (1981) and Mang et al. (1982). Several methods have been used to extract RNA from SBMV. For instance, lithium chloride has been used to prepare RNA and coat protein for studies on virus assembly (Savithri and Erickson, 1983). Phenol-chloroform extraction has been used by Rutgers et al. (1980), Salerno-Rife et al. (1980), Ghosh et al. (198 1) and Manget al. (1982) to prepare RNA for in vitro translation, and by Veerisetty and Sehgal (1980) to prepare RNA to study the effect of proteinase K on infectivity. In all methods, the

* To whom correspondence 016h-09~J/X5/S0.i.?0

should

be addressed

c 19X5 Elhevler Science Publishers

B.V. (Biomedical

LX\ision)

structure of the capsid has to be relaxed before dissociation of the virions and separation of the RNA from coat protein. As Jean and Sehgal (1976) found that different methods gave RNAs of different infectivities, we investigated the extent to which methods extent,

the choice

of method

were used to extract reproducibility,

MATERIALS

affected

the in vitro

translation

RNA and in this paper we report

and products

of translation

of the RNA.

Four

the effects on the

in vitro.

AND METHODS

Whenever possible, aqueous minimum of 20 min.

solutions

and glassware

were sterilised

at 120°C for a

Propagation and preparation of virus The cowpea strain of SBMV was propagated as described by Hull (1977). Virus was purified by the method of Denloye et al. (1978) with the following modifications. (1) Before polyethylene glycol (PEG) precipitation, Triton X-100 was added to 1% (v/v). (2) The final high-speed centrifugation was omitted. The virus was pelleted from 25 ml of suspension through a 5 ml cushion of 25% (w/v) sucrose in 0.05 M sodium acetate pH 5.0 by centrifugation at 27,000 revolutions per minute (rpm) for 3.5 h in a Beckman Type 30 rotor at 4°C. (3) The pellet was resuspended in0.05 M sodium acetate pH 5.0, and caesium chloride was added to give a final density of 1.36 g/ml. After centrifugation (Beckman Type 40 rotor, 36,000 rpm, 17.5 h, ISOC), the virus band was removed and dialysed against several changes of 0.05 M sodium acetate pH 5.5. The final suspension contained 3.3 mg virus/ml, based on an E&j,% = 5.85 (Tremaine and Hamilton, 1983). Extraction of RNA Approximately 0.8 mg of SBMV was precipitated from 10% (w/v) PEG, 1% (w/v) NaCl and pelleted (Sorvall SS-34 rotor, 8,000 rpm, 10 min, 4°C) in each of 12 silicone-coated Corex(Corning Glassworks, U.S.A.) tubes. Each pellet wasresuspended in 0.5 ml of glass distilled water, and the A 26Owas recorded for the calculation of the theoretical yields of RNA. Dissociation buffer(DB) was 2% (w/v) sodium dodecyl sulphate (SDS), 2 mM EDTA, 0.2 M Tris-HCI pH 8.25; this has been reported to dissociate SBMV within a few minutes (Sehgal, 1973). Four methods were used to separate the RNA from coat protein. Each method was applied to three samples of virus. (I) Sucrose density gradient centrifugation (Sehgal, 1973). An equal volume of DB was mixed with the resuspended virus. The tubes were stored at 5°C for 1 h and their contents layered on linear 8-40% (w/v) sucrose gradients (Serva: pyrogen, RNA- and DNA-free sucrose) containing 0.02 M sodium acetate pH 7.0, and 0.1% SDS. After centrifugation (Beckman Type SW 27 rotor, 23,000 rpm, 16 h. 20”C)thegradientswere

197

passed through

an ISCO Model 640 Density Gradient

254 nm was monitored, were collected.

and the fractions

containing

This RNA was precipitated

overnight

of cold 96% ethanol,

4% 3 M sodium

10,000 rpm, 10 min, 4°C)

and washed

acetate

Fractionator,

the absorbance

at

the major peak of absorbance at -20°C by adding

pH 5.5, collected

(Sorvall

2.5 volumes SS-34 rotor,

by reprecipitation.

A 1: 1 mixture of chloroform (2) Phenol-chloroform extraction (Zimmern, 1975). and water-saturated phenol was pre-saturated with an equal volume of half-concentration DB. DB (0.5 ml) was added to the virus suspension, and the tubes were shaken for 10 min at room temperature. One ml of the phenol-chloroform mixture was added to each tube, and the contents were whirlimixed for 10 min. Phases were separated by centrifugation (Sorvall SS-34 rotor, 8,000 rpm, 10 min, lS”C), and the organic phase was re-extracted with 1 ml of half-concentration DB. The aqueous phases were pooled and re-extracted with 2 ml of phenol-chloroform, rinsed three times with 2 ml of fresh, cold, peroxide-free ether, and the remaining ether was removed using nitrogen. RNA was precipitated and washed as above. (3) Sodiumperchlorate

extraction (Wilcockson

An equal volume of and Hull, 1974). 10% SDS was added to the virus suspension. The mixture was kept at 60°C for 5 min, and washed using 3 ml of 8 M NaClO, into a nitrocellulose tube. Centrifugation (Coolspin 1Zplace universal swing-out rotor, 2,200 rpm, 10 min, 5°C) caused the denatured protein to form a raft. The tube was punctured to remove the liquid, and the RNA was precipitated and washed as above. (4) Lithium chloride precipitation of RNA (Savithri and Erickson, 1983). One volume of DB was added to the virus suspension, and each tube was stored on ice for 1 h. One ml of 4 M LiCl in 0.01 M Tris-HCI, pH 7.0, was added and the tubes were frozen overnight at -20°C. After thawing, the precipitate was collected (Sorvall SS-34 rotor, 10,000 rpm, 10 min, 5°C) and redissolved in water. The lithium chloride, freezing, pelleting, and dissolving as described above.

steps were repeated,

and the RNA was washed using ethanol,

In vitro translation

The mRNA-dependent rabbit reticulocyte cell-free lysate (MDL) was prepared according to Pelham and Jackson (1976). It contained 3 1 mM potassium and 1.7 mM magnesium (measured by flame atomic absorption spectroscopy). One sample from each of the 12 RNA preparations was translated in each of two independent tests. Each 20 ul incubation cocktail contained 40% (v/v) MDL, 112 mM K’, 0.8 mM Mg2+ (acetate salts were added to supplement endogenous levels), 10 mM creatine phosphate, 0.5 mM dithiothreitol (DTT), 5 uCi of L-[35S]methionine, all other essential amino acids at 0.05 mM, and 1.2 ug of SBMV RNA. After incubation at 30°C for 110 min, a 3 ul sample was removed from each cocktail to assess the incorporation of

198

radioactivity

into trichloroacetic

son, 1976). An equal volume

acid (TCA)-precipitable

material(Pelham

and Jack-

of sample buffer (10% SDS, 0.5 M DTT and 20% (v/v)

glycerol, in 0.4 M Tris-HCl, pH 6.8) was added to each cocktail. Samples were then boiled for 3 min. “C-labelled marker proteins (Amersham International, plc), and incubation samples containing equal counts per minute (cpm) of TCA-precipitable material were made up to unit volume with 40% MDL and electrophoresed on a 15% (w/v) polyacrylamide gel containing SDS (Laemmli, 1970). Labelled bands were visualised by fluorography and exposure to Fuji RX X-ray film at -70°C. Statistical tests Siegel (1956) gives details

of the tests used in this paper.

RESULTS

Various properties and are summarized

of SBMV RNA, extracted in Table 1.

by the four methods,

were measured

(I) Yields Theoretical yield was calculated assuming recovery of all RNA, and that the virus is 21% RNA by weight (Ghabrial et al., 1967). The mean yield of each method differed significantly from that of any other method (2-tailed randomisation test, P
TABLE

I

Properties

of SBMV RNA extracted

Treatment

Percentage

dissociation

Mean (range)

by

yield

various

treatments

Stimulation

Ax,,/Axo Mean (range)

protein

Mean (ranec)

and then: Sucrose

of

synthesis”

density

gradient

73 (62-84)

1.90 (1.8X-1.93)

29.8 (27.7-32.0)

85 (77-94)

I .94 ( I .X7-2.07)

33.9 (31.5-39.4)

14 (7-22)

1.75 (1.35-2.15)

10.8 (10.2-I

39 (3 l-43)

I .99 ( I .96-2.02)

31.2 (23.6-38.8)

centri-

fugation Phenol-chloroform extraction Perchlorate

1.9)

precipitation Lithium

chloride

DreclDitation a The stimulation

of protein synthesis

given RNA + cpm incorporated

in an incubation

in incubations

wascalculated

containing

as: cpm incorporated

no exogenous

mRNA.

In response to a

199

order lithium

of increasing chloride

chloroform

yield,

extraction,

the methods sucrose

are: perchlorate

density

gradient

precipitation centrifugation

of protein, and phenol-

extraction.

(2) Purity of RNA Freedom from protein was assessed by the A 260/A280 ratio. The mean purities of RNA prepared by each method were compared. Sucrose density gradient centrifugation differed significantly from lithium chloride extraction (randomisation test, P
free from protein.

(3) Stimulation of protein synthesis during in vitro translation Total protein synthesis was measured by the amount of radioactivity incorporated into TCA-precipitable material. RNA extracted by the perchlorate method stimulated protein synthesis IO-fold. This was significantly (Mann-Whitney U test, P
of of

relative molecular masses 94 X 10’ (94K), 63K and 29K presumably correspond to the CPl (lOOK), CP2 (70K) and CP3 (30K, identified as the coat protein) respectively of Mang et al. (1982). Fig. 1 also shows minor virus-specific products of 57K, 42K, 31K and 30K, and an endogenous reticulocyte band of 46K. The band of 46K frequently appears in incubations programmed with water alone or with a wide variety of messenger RNAs (data not shown). This band is thought to be an artefact of the reticulocyte lysate system. However, the possibility of an additional SBMV-specific product

of this size cannot

be excluded.

The method of RNA extraction affects the pattern and reproducibility of polypeptide products. Only one preparation of perchlorate-extracted RNA gave discrete bands in a recognisable pattern. In all other methods, the pattern of polypeptide products does not vary between individual RNA preparations. The perchlorate-extracted RNA also produced a set of polypeptides somewhat different to that produced by RN.4 from the other three methods. The products of the other three methods are quite similar to each other, but there are qualitative and quantitative differences. CPl (94K) is only produced by phenol-chloroform or by lithium chloride-extracted RNA.

cl3 l-

63, 57, 46, 42,

31, 30, 29r

Fig. I. I.-[‘S]Methionine-labelled sucrose

density

gradient

rate precipitation

(tracks

marker

proteins

material

of RNA. (Amersham

major SBMV RNA-coded

of in vitro translation

(tracks

7-9) or lithium chloride

cpm of TCA-prrcipitable dent preparations

products

centrifugation

Track

from rabbit

precipitation(tracks reticulocyte

polypeptides

extraction

plc). The postttonsand

independently

(tracks 4-n)

10-12). Each trackcontained

lysate incubations

13: 7.200 cpm of endogenous

International,

of RNAs prepared

l-3), phenol-chloroform

programmed

lysate products,

Track

relative molecular

by:

perchlo75,000

with indepen14: “C-labelled

masses (X IO-‘)ofthe

are shown on the left and those of 14C-labelled marker

proteins

on

the right.

The proportion chloroform prominent

of CP3 (29K) varies.

It is the most prominent

extracted or sucrose gradient-fractionated RNA, products of lithium chloride-extracted RNA.

product

of phenol-

but one of the least

DISCUSSION

We have shown that different methods of extraction give different yields of RNA, and that there are differences in the purity and in vitro translation properties of the RNAs. SBMV RNA prepared by perchlorate extraction gave the lowest stimulation of protein synthesis. Only one of the three RNA preparations gave discrete products, and overall, the pattern appeared more heterogeneous than those from other RNA preparations. We conclude that this extraction method is inefficient and unreliable. Our

201

yields were at best 22%. However,

Wilcockson

and Hull (1974) and Jean and Sehgal

(1976) have reported yields of 50% and 33% respectively. The poor translation in vitro of perchlorate-extracted RNA may have been due to contamination by protein. Also, perchlorate-extracted suggesting

RNAs

that the templates

gave smears

of lower

molecular

mass polypeptides,

were fragmented.

The other three methods gave consistently better results. Ideally, the method of choice should give polypeptide products as similar as possible to those found in vivo. However, in the absence of information on SBMV gene products in vivo, the most useful method is phenol-chloroform extraction. It gave the highest yields of relatively pure RNA which stimulated protein synthesis quite well, and gave a full range of discrete polypeptide products. Mang et al. (1982) showed that full-length RNA produced CPl (lOOK), CP2 (70K) and CP4 (20K), whereas a smaller subgenomic species produced CP3 (30K). As noted, the method of RNA extraction affected the proportion of CP3 produced. This suggests, that the RNA samples differed in the amounts of each species they contained. These observations highlight the need for care when deciding how to prepare RNA for in vitro translation. ACKNOWLEDGEMENTS

We wish to thank T. Davies, R. Falks and M. Gee, of the A.F.R.C. Food Research Institute, Norwich, for performing the atomic absorption spectrophotometry. M. Hobbs typed the manuscript, and Dr. E. Arthur advised on statistical tests. M. Brisco holds a Postgraduate Studentship from the Department of Education for Northern Ireland. REFERENCES

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