Punjabi HD Movies | Ant-Man and the Wasp 2018 720p BluRay H264 AAC-RARBG | Wrecker (2015) [ENG]

In vitro synthesis of chloroplast ferredoxin as a high molecular weight precursor in a cell-free protein synthesizing system from wheat germs

In vitro synthesis of chloroplast ferredoxin as a high molecular weight precursor in a cell-free protein synthesizing system from wheat germs

Vol. 82, No. 4, 1978 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 29,1978 Pages 1121-1131 IN VITRO SYNTHESIS OF CHLOROPLAST FERREDOX...

874KB Sizes 0 Downloads 0 Views

Vol. 82, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 29,1978

Pages

1121-1131

IN VITRO SYNTHESIS OF CHLOROPLAST FERREDOXIN AS A HIGH MOLECULAR WEIGHT PRECURSOR IN A CELL-FREE PROTEIN SYNTHESIZING SYSTEM FROM WHEAT GERMS J.G. +

Huismanf

Department Amsterdam,

A.F.M.

Moormanx

and F.N.

of Plant Physiology, IJdijk 26, Amsterdam,

Verkley+

University of The Netherlands

XSection for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Eerste Constantijn Huygenstraat Amsterdam, The Netherlands Received

April

20,

20,1978

Summary: RNA isolated from leaves of Nicotiana tabacum, etiolated leaves of Phaseolus vulgaris and from cells of Chlamydomonas reinhardii, can be translated in cell-free extracts derived from wheat germs. The in vitro products were identified by SDS electrophoresis after imTn=ipitation with specific antisera against ferredoxin. The in vitro products appeared to have a Mr 20,500, in contrast to authentic ferredoxin that has a Mr 17,000. We propose that chloroplast ferredoxin is synthesized in vitro as a biosynthetic precursor. Ferredoxin synthesis was ?&to be associated with poly(A)-RNA, which supports our previous conclusion that ferredoxin messenger is a transcript from the DNA of the nucleus. Ferredoxin

plays

chloroplasts presence candidate

for

in

of virtually in both

genetic

a role

all

prokaryotes

to be encoded studies

in nuclear

sized

on cell-sap

plast

is

not

To learn

photosynthetic

organisms

in chloroplast

have

DNA. Thus

and is

and eukaryotes

for

(2,3,4)

photosynthesis

it

ribosomes,

revealed is

likely

although

makes

found

it

in

(1).

the Its

an attractive

DNA. However,

recent

that

ferredoxin

is

encoded

that

ferredoxin

is

synthe-

mRNA import

into

the

chloro-

excluded. more

about

the

molecular

events

underlying

Abbreviations: SSC: 0.15 M NaCl, 0.015 M Na-citrate Fd: ferredoxin; PBS: 140.4 mM NaCl, 9 mM Na HPO4, (pH 7.4); CTA-Br: cetyl trimethyl ammonium i4 romide

chloro-

(pH 7.0); 1.3 mM NaH2P04

0006-291x/78/0824-1121$01,00/0 1121

Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 82, No. 4, 1978

plastal

biogenesis

chloroplast In under

this

paper

direction

findings

we have

ferredoxin

P.vulgaris

in

on the

possible

the --in vitro

origin

products

in

of precursor regulation

synthesis from

RNA from

of

the

for

wheat of

germs.

ferredoxin

N.tabacum,

confirms

gene

of

our

previous

ferredoxin

the

immunoprecipitate

gel

electrophoresis.

Ferredoxin

molecular

precursor.

as a high this

vitro

synthesis

The result

nuclear

of

--in

RESEARCH COMMUNlCATlONS

system

poly(A)-containing

red to be synthesized The implications

the

a cell-free

by SDS-polyacrylamide

processing

studied

we report of

AND 8lOPHYSlCAL

and Chl.reinhardii.

The translation rized

BIOCHEMICAL

result

ferredoxins

by chloroplast

with

weight respect

(c.f.

5,6,7)

factors

are

(3,4).

were characte-

to questions

appea-

of

and their discussed.

METHODS: Seeds of Nicotiana tabacum were germinated in Perlite on a synthetic medium (8) in a conditioned room under a 12 h photoperiod of 5000 lx white light at 20 + 2'C. Seeds of Phaseolus vulgaris were germinated under the same conditions but kept in the dark. Before the extraction of RNA these plants were illuminated for 1 h at 5000 lx white light. Chlamydomonas reinhardii (wt) was grown in liquid medium according to Surzycki (9). Ferredoxin: Ferredoxin was isolated and purified as described previously (3). Preparation of RNA: About 10 g de-veined lo-day-old leaves or packed cells of Chlamydomonas were first frozen in liouid nitroqen and then pulverized carefully into suspension in sterile 10 mM The cold suspension was then dissolved in an Tris-HCl (pH 7.5). equal amount of a mixture containing 2% SDS, 1% tri-iso-propylnaphtalene-disulphonate, 2% sodium-p-amino salicylate and 6% secbutanol. After filtration through cheese-cloth, RNA was extracted using the phenol/cresol method modified by Moorman et al.(lO) and precipitated by addition of l/lOth volume 3 M Na-acetate (pH 5.5) and 2.5 volume ethanol. All RNA preparations were treated with DNA-ase I (ll), pronase (11) and CTA-Br (12). The phenol/cresol step and the ethanol precipitation was repeated twice. Electrophoresis of RNA preparations: Gel electrophoresis of RNA was carried out as described by Peacock and Dingman (13). Isolation of poly(A)-RNA: RNA preparations, approximately 1 mg, were pelted out by incubation in O.lxSgC and 0.1% SDS for 10 min at 60 C and, then rapidly chilled at 0 C. After raising the salt concentration to 0.4 M NaCl in 0.1 M Tris-HCl, 2 mM EDTA and 0.2% the RNA was applied to a poly(U)-Sepharose 4B column SDS (gH 7.5), at 20 C and fractionated by lowering the NaCl concentration from 0.4 M to 0.01 M. Poly(A)-RNA was extracted from the column with 90% formamide in 10 mM K-phosphate and 2 mM EDTA (pH 7.5) (14). After fractionation the RNA was precipitated with 2.5 volume res ecethanol, washed several times in cold 70% and 96% ethanol, tively, dried and dissolved in sterile water and stored at -80 8 C until used.

1122

Vol. 82, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cell-free protein synthesizing system from wheat germs: p5S], Methionine or ?Hf-labeled amino acids (Amersham-Searle, UK) were incorporated into protein using a cell-free extract from wheat germs assayed essentially as described by Roberts and Paterson (15). The S23 extfactq were prepared according to Marcu and Dudock (16). Optimal Mg* , K and RNA concentration was 3.5 mM, 125 mM and 800 ug/ml, respectively. Protein synthesis activity from a 5 ~1 sample was measured as hot TCA insoluble radioactive products (17). The fidelity of translation was determined in some experiments, using Alfalfa mosaic virus RNA (18). Immunological assay of ["S]-labeled ferredoxin: Protein synthesis assays with a volume of 75 ~1 were stopped by adding 5 ~1 250 mM Na-EDTA (pH 7.5) and 20 ul of a solution containing 250 mM NaCl, 250 mM NaPi (pH 7.2), 5% Triton X-100 (w/v), 2.5% deoxycholate (w/v), 0.5% SDS (w/v) and 5 mM methionine. The reaction mixture was then centrifuged at 100,000 x g for 20 min at 5 C. The supernatant was used for immunoassays. It was divided in two and each part reacted with antibodies raised against ferredoxin (4) and pre-immune serum, respectively. The complexes were precipitated by addition of goat anti-rabbit-IgG (kindly supplied by Dr. L.A. Grivell, University of Amsterdam) and prepared for measuring the radioactivity. The entire immunological assay used was as described in more detail by Moorman et al. (10). Qualitative analysis of [3sS1-labeled ferredoxin: Labeled products were subjected to electrophoresis in 13.5% polyacrylamide gels in the presence of SDS according to Laemmli and Favre (19). The immunoprecipitates were dissolved in sample mixture containing 12.5% SDS (w/v) , 25 mM dithiothre&tol, 20% glycerol (w/v) and 8 mM urea and incubated for 2 h at 37 C. Samples were boiled for. 2-5 min before being applied to the gels. After electrophoresis gels were stained with Coomassie blue, destained and prepared for fluorography with dimethylsulfoxide and diphenyloxazole (20), dried and exposed with Kodak RP-OXOMAT medical X-ray film, preflashed with red light, and supplied with an Ilford X-ray intensifying screen. '2510dinated ferredoxin of N.tabacum was used as radioactive marker. Iodination was with 3 rnq bferredoxin (3) dissolved in 0.5 ml PBS and incubated with 0.5 mCi 125iodine (Amersham-Searle, UK) and 100 pg chloramine-T (21). Iodinated ferredoxin was recovered by fractionation on Sephadex G-50 coarse column. RESULTS: leaves

similar 18S,

Loening

derived in

and Ingle

Template Phaseolus, results

Fig.

13s and 4s.

ribosomes

This from

RNA preparations and cells

isolated

in agreement

cytoplasmic, leaves

from

of Chl.reinhardii

1 shows distinct

is

greening

RNA bands with

the

25S,

diversity

mitochondrial and algae,

at

of

and chloro-

as determined

by

(22).

activity Nicotiana

in

of

and P.vulgaris

results.

RNA compunds plast

gels

of N.tabacum

yielded 23S,

polyacrylamide

a stimulation

of

RNA preparations:

and Chlamydomonas of

to

[ 3 S~]-methionine 1123

Addition

of

RNA from

a wheat

germ

system

incorporation

into

BIOCHEMICAL

Vol. 82, No. 4, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from 2.4% polyacrylamide gels of RNA prepaFig. 1. Densitograms rations prepared from leaves of P.vulgaris (A), N.tabacum (B), and cells of Chl.reinhardii (C). S-values are given relative to the mobility ofE.coli rRNAs (215 and 16S, respectively). --

protein

of

initial

experiments

added if

plant

the

both

feature

messengers. ing

purified

of the

respectively

germ for

system

this

by the

with

will

RNA was drastically

Unfractionated,

between

separated

rJ.tabacum

fractions bound

from

Fig.

2:

then

the

tem-

found

(23,24,14).

chloroplast RNA into

and nonbound

activity

to be We used

and cell-sap poly(A)-contain-

by poly(U)-Sepharose

1124

to

Template

RNA is

RNA fractions

to discriminate

In

reduced.

RNA fractions:

poly(A)-lacking

2).

weakly

be clear treatment

RNA and mitochondrial

We have

(Fig.

responded

CTA-Br

of Poly(A)-containing

and poly(A)-lacking

tography.

wheat

The reason

chloroplast

associated

40 and 35-fold, the

RNAs.

activity Isolation

this

90,

RNA was not

plate

of

about

RNAs were

4B chromatested

for

Vol. 82, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

I L-4-l

03

unfractionatedt

0.4 O.~ a., 0.05 0.01 0 MNaCl : 4 Formamide

Fig. 2. Characteristics of RNA directed translation in a cell-free system from wheat germs. Stimulation of the protein synthesis was measured by CS5S]-methionine incorporation into hot TCA precipitarate is given as a funcble material per 75 ~1. The incorporation tion of the concentration of RNA from: P.vulgarisA-A; Chl.reinhardii 0 -0; N.tabacumn0; N.tabacum not treated with K-Brm -m. See Methods for the condityon of the assay. [35S]-methionine (22,9 nmol)(65 uCi/ml). Incubation time was 90 min at 200C. The values are corrected for endogeneous incorporation Values are the means of triplicate experiments. (5,500 cpm/75 pl). Fig. 3. Fractionation of total RNA prepared from leaves of N.tabacum by poly(U)-Sepharose 4B chromatography. Each bar repreRNA was precipitated as described in Zents a 3 ml fraction. Methods and added to a wheat germ system. Protein synthesis was measured by ["S]-methionine incorporation into hot TCA precipitable material per 5 ~1.

their

ability

hot

to stimulate

TCA precipitable

template

activity

fractionated

is

over

the

Immunological

ability

Figure

associated

with

endogeneous

identification

RNA preparations

and poly(A)-RNA

isolated

to stimulate

-methionine

products.

and some nonbound

stimulation

Total

["S]

3 shows that the

most

into of the

[email protected], about

un-

two-fold

level. in

vitro

N.tabacum-RNA,

incorporation

of

1125

synthesized

Phaseolus

Nicotiana,

from

bound

RNAs show only

of

from

incorporation

were

L3'~]-methionine

ferredoxin:

and Chlamydomonas tested

for into

their ferre-

Vol. 82, No. 4, 1978

Table

BIOCHEMICAL

1. IMMUNOPRECIPITATION

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

FROM [3S~]-~~~~~~

PRODUCTSOF

PROTEIN SYNTHESIS IN A CELL-FREE SYSTEM FROM WHEAT GERMS

RNA used for in vitro protein Synms

input per immunoprec.

total total

240.236 479.455

RNA P.vulgariS RNA E.vulgaris

percentage bound pre-immune IS 0.90 1.42

radioactivity by anti-Fd I+ 2.00 2.78

net value 1.10 1.36

total RNA N.tabacum total RNA E.tabacum poly(A)-RNE N.tabacum

238.558 198.222 148.222

1.08 1.04 0.98

1.24 1.20 1.58

0.16 0.16 0.60

total total

200.890 160.664

0.88 1.20

1.08 1.40

0.20 0.20

total

RNA C.reinhardii RNA c.reinhardii

RNA N.tabacum

anti-fungal cell-wall protein IgG -0.03 0.98

1.01

200.040

Stimulation of the protein synthesis was measured by ['"s]methionine incorporation into hot TCA precipitable material per 5 ill. Results are given from two separately isolated RNA prepaFor conditions of protein synthesis rations from each species. and immunoprecipitations see Methods and the Legends to Fig. 2,3.

doxin

--in

vitro.

The labeled

immunological

test

tabacum-Fd-IgG ferredoxin raised cript

against

to higher acid

than

species

acids.

been

were

As clear

with

total

RNA are serum

of Chlamydomonas detected

from

2 a low but

pre-immune

serum.

1126

1).

presence

by specific The lack

Since is

of

reproducible

reactivity

the

the

['HI-labeled

anti-Fd-IgG, of

manus-

unknown

ferredoxin,

in

was bound

the

et al.

ferredoxin

in Phaseolus

Table

Antibodies

products --In vitro bound by anti-Fd-IgG

(Table

translated

of radioactivity

comparison

of

(4).

(J.G.Huisman similarly.

by pre-immune

composition has not

of these

amount

direction

selected

anti-!.

and P.vulgaris

previously

ferredoxin

to a specific

purpose,

to N.tabacum

as described

were

subjected

For this

binding

Chlamydomonas

extent

methionine

amino

selected

under

were

ferredoxin.

strong

preparation)

synthesized

amino

with were

in

for

products

higher in of -in

and RNAs

Vol. 82, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table 2. IMMUNOPFE~IPITATION FROM [3~]-LABELED PRODUCTS OF PROTEIN SYNTHESIS IN A CELL-FREE SYSTEM FROM WHEAT GERMS RNA used for in vitro protein SynZiZZs total

input per immunoprec.

percentage bound pre-immune IgG 0.54 1.17

radioactivity by anti-Fd IgG 1.14 2.88

net value 0.60 1.71

total

RNA P.vulgaris RNA P.vulgaris

120.444 684.676

total total

RNA C.reinhardii RNA C.reinhardii

101.880 154.275

0.74 0.75

1.08 1.05

0.34 0.30

total

RNA N.tabacum

264.880

0.88

1.06

0.18

See Table 1. foralanine details ( 3; ~;~~~y;~ug~~~o ,;;y;,=;, yrginine (11 Ci/mmol); lysine (33 Ci/mmol); tyrosine (43 Ci/mmoi) and valine (32.6 Ci/mmol): 1 mCi/ml for each amino acid, respectively.

vitro

products

proteins,

with

implies

Fd-IgG

is

the

for

poly(A)-RNA

whereas

product

for

0.16%

ferredoxin

from (Table

evidence

against

fungal

iaununoprecipitation

synthesized

can be bound

by anti-Fd-IgG

This

for

the

nuclear

--In

0.6%,

can be bound enhancement

RNA. We consider

origin

of

vitro

direction

for

a 3-4-fold

to total

anti-

1).

under

N.tabacum-RNA

indicates

relative

using

(Table

polypeptides

1).

cell-wall

test

ferredoxins

unfractionated

synthesis

to be strong

raised

chloroplast

["'S]-labeled

of N.tabacum

of

that

specific

synthesized

only

antibodies

this

ferredoxin

messen-

gers. Electrophoretic doxin:

Total

direct

the

characterization RNA and poly(A)-RNA

synthesis

of

Ferredoxin

was isolated

The entire

immunoprecipitates

subjected ferredoxin.

of

to SDS gel

ferredoxin

of

synthesized

ly.tabacum

were used

wheat

indirect

the

germ

in

and compared

large the

1127

amount

ferreto

system.

immunoprecipitation.

were dissolved

electrophoresis

radioactivity

vitro

in the

by means of

To accomodate

and low amount

from

in

of

electrophoresis

sample with

protein

mix

and

authentic (mainly

IgG)

was performed

Vol. 82, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

04

05

of N.tabacum ferredoxin synthesized by Fig. 4. Characterization total RNA and poly(A)-RNA of N.tabacum in a cell-free protein synthesizing system from wheat germs. Analysis was by SDS slab-gel electrophoresis and fluoroqraphy. FLUOROGRAM: (track 11, [" I]-Fd-N.tabacum-IgG-complex; (track 21, t"SJ-methionine product of total-RNA, immunoprecipitated with immunoprecipitated with anti-Fdproduct of poly(A)-RNA, %munoprecipitated with pre-immune IgG; (track 51, as 4, immunoprecipitated with anti-Fd-N.tabacum-IgG. Markerproteins: bovine ser%nnn (68,000); ovalbumin (43,000); cytochrome C (12,400); trypsin inhibitor (6000). of P.vul aris and Chl.reinhardii ferreFig. 5. Characterization doxin, synthesized by their own in cell-free protein---p-7poly(A-RNA synthesizing system from wheat germs. Analysis was by SDS slab-gel electrophoresis and fluoroqra hy. FLUOROGRAM: (track 11, [' 51~-Fd-N.tabacum-IgG-complex; (track 2), P'SJ-methionine product of poly(AT-RNA of N.tabacum, immunoprecipitated with anti-Fd-N.tabacum-IgG; (track 3), as 2, poly(A)-RNA of P.vulgaris; (track 4),, poly(A)-RNA of Chl.reinhardii, immunoprecipitated with anti-Fd-Chl.reinhardii-m. Calculations of the Mr same as forFig. 4.

in gels

of

3 mm thickness.

Approximately

0.01

IgG

as described

identical

Figure

4 shows the

serum

reacted

(Fig.

4, track

[lz51]-Fd

pg [125 I]-Fd

results

was used as marker

was allowed

in methods obtained

with

for from

to react --in vitro specific

with

anti-Fd-

products. and pre-immune

["S]-labeled polypeptides. --in vitro 1) has the same electrophoretic mobility

1128

protein.

[1251]-Fd as authen-

Vol. 82, No. 4, 1978

tic

BIOCHEMICAL

ferredoxin

and migrates

be calculated but

from

probably

prevents fluorogram

were

which

the

-about gel

weight

precursors

DISCUSSION: in

This

from

We have

from

is probably

which

is concomitant

To discriminate fractionated affinity

having

short

Moreover,

the

much more

efficiently

cordantly

we found

thesis

it

between

chloroplast

wheat

much better

than that

the

of

it molecular

from wheat was the

to the

translates bacterial

(27). messengers,

1129

3) than

we RNA,

of chloroplast

RNAs

poly(U)

cannot

eukaryotic

species

active,

development,

column

concentration

type

germs.

most

RNA synthesis

at low NaCl

Fig.

in

can be trans-

and cell-sap

bind

poly(A)-RNA

(20-fold,

of

ferredoxin

of plastid

The existence

system

The bands

and poly(A)-containing

that

germ

preci-

weights

system

onset

poly(A)-lacking

unlikely

again

as higher

of Phaseolus

an increase

is

from

RNA.

induced

poly(A)-tracks but

be excluded,

leaves

chromatography.

experi-

RNA preparations

with

RNA into

using

translated

plant

due to light

1?hen the

may be overestimated

synthesizing

etiolated

Mr

authentic

poly(A)-containing

protein

protein,

molecular

value

binds

contrast,

(see Fig.5).

than

are

shown that

a cell-free

RNA isolated

greater

ferredoxins

In

translated

Mr 20,500

this

serum

, anti-Fd-IgG

have apparent

(2,3),

2 and 4 on the

products.

polypeptides

can

which

pre-immune

anti-Fd-IgG.

Even though

12,000

Tracks

that

poly(A)-RNA

than

ferredoxin,

with

daltons

that

of

radioactive

labeled

products

system.

i.e.

a single

peptide,

3-4000

suggests

lated

indicates

contain

is greater

composition

character

was precipitated

--in vitro

This

in discrete

a radioactive

20,500

still

which

using

RESEARCH COMMUNICATIONS

of SDS (25,26).

and Chlamydomonas

pitated

this

empty

was repeated

Phaseolus

from

binding

3 and 5 both

20,500,

acid

acidic

no radioactivity

tracks

ment

proper

at Mr 17,000.

amino

due to the

the

virtually

its

AND BIOPHYSICAL

(14,23,24) messengers

of mRNA (28). stimulate

poly(A)-lacking

Con-

protein

Syn-

RNA

Vol. 82, No. 4, 1978

species al.

BIOCHEMICAL

This

do.

is

is

assumed

and anti-Fd-IgG

that is

precipitation

peptides

with

than

it

the

findings

that

of Bottomley

weight

mean that

it

IgG,

--in

et

is

carboxylase,

precursor.

the

--in vitro

native

protein

follows

a similar

incorporation

of

chloroplast,

since in

the

the plastid

into

the

iron

native

storage

we

The fact eviden-

DNA and not

by

has a higher by us to

must

yet

to be

of Ribulose-l,S-bissynthesized

molecular

precursor

We suggest scheme,

that

earlier

protein

this

of the

product

as a high

(6,7).

most

ferredoxin.

this

chloroplast

chloroplast

translated

interpreted

sub-unit

small

biosynthetic iron

is While

synthesized

the

the

ferredoxin

Thus

vitro

our

that

another

also

of

by nuclear

IgG

as evidence

supports

precursor.

Entering

release

translation

for

the

--in

that

coded

native

that

the

fact

pre-immune ferredoxin.

is taken

poly(A)-RNA

ferredoxin

is

to bind

and the

vitro

from

than

is

cytoplasm,

localized

the

is clear

phosphate

ability

as one peptide,

ferredoxin

molecular

between

of more of

DNA. The finding

it

difference

specific

translated

chloroplast

only

by pre-immune

studying

is

ce (3,4)

proved,

the

migrates

have been that

the

by anti-Fd-IgG

radioactivity

the

consistent

(29.) It

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and that protein,

protein:

weight is

that

in

cleaved

to

ferredoxin the

last

occurs

in

step,

the

the

phytoferritine,

is

(30).

Acknowledgments: We gratefully acknowledge the expert technical assistance of MS M.G.Th. Gebbink. We are indebted to Dr. A.Mussrave and Prof.Dr. D.Stegwee for critical reading the manuscript. Wethank Prof.Dr. G.S.P.Groot for constructive discussion. This work was supported in part by The Netherlands Organization for the Advancement of Pure Research (ZWO) grant nr: 86-35. REFERENCES: 1. Buchanan, 2. Kwanuyen, 405,

B.B., and Arnon, P., and Wildman,

D.I. S.G.

(1970)

(1975)

Adv. Enzym. 33, 119-176. Biochem. Biosys. Acta.

167-174.

3. Huisman, J.G., Gebbink, M.G.Th., Modderman, (1977) Planta 3, 97-105. 4. Huisman, J.G., Bernards, A., Liebregts, P., and Stegwee, D. (1977) Planta E, 289-296.

1130

P.,

and Stegwee,

Gebbink,

M.G.Th.,

D.

Vol. 82, No. 4, 1978

5. Blobel,

G.,

8lOCHEMlCAL

and Dobberstein,

AND BIOPHYSICAL

B.

(1975)

RESEARCH COMMUNICATIONS

J.

Cell

Biol.

67,

835-851.

6. Dobberstein, B., Blobel, G., and Chua, N.H. (1977) Proc. Nat. Acad. Sci. 74, 1082-1085. 7. Highfield, P.E., and Ellis, R.J. (1978) Nature 271, 420-424. 8. Sluiters-Scholten, C.M.Th., Van den Berg, F.M., and Stegwee, D. (1973) Z. Pflanzenphysiol. 69, 217-227. 23, 67-73. 9. Surzycki, S. (1971) Methods Enzymol. 10. Moorman, A.F.M., Grivell, L.A., Lamie, F., and Smits, H.L. (1978) Biochem. Biophys. Acta. in press. 11. Van Ommen, G.J.B., Groot, G.S.P., and Borst, P. (1977) Mol. Gen. Gen. 154, 255-262. P.R. (1966) Arch. Biochem. Biophys. 12. Spencer, D., and Whitfeld, 117, 337-346. 13. Peacock, A.C., Dingman, C.W. (1967) Biochemistry 5, 1818-1827. 14. Groot, G.S.P., Flavell, R.A., Van Ommen, G.J.B., and Grivell, L.A. (1974) Nature 252, 167-169. 15. Roberts, B.E., and Paterson, B.M. (1973) Proc. Nat. Acad. Sci. 2, 2330-2334. 16. Marcu, K., and Dudock, B. (1974) Nucl. Acid. Res. I, 1385-1397. 17. Moorman, A.F.M., Lamie, F., and Grivell, L.A. (1976) Febs Letters 7l, 67-72. 18. Van-Vloten-Doting, L., Rutgers, T., Neeleman, L., and transcription and translation of Bosch, L. (1975) In vitro viral genomes, pp. 233-242, Colloque INSERM, Paris. Biol. 80, 575-599. 19. Laemmli, U.K.,and Favre, M. (1973) J. Mol. 20. Laski, R.A., and Miles, A.D. (1975) Eur. J. Biochem. 56, 335-341. 21. Hunter, W.M., and Greenwood, F.C. (1962) Nature 194, 495-496. 22. Loening, U.E. and Ingle, J. (1967) Nature 215, 363-367. 23. Sagher, D., Grosfeld, H., and Edelman, M. (1976) Proc. Nat. Acad. Sci. 2, 722-726. 24. Wheeler, A.M., and Hartley, M.R. (1975) Nature 257, 66-67. 25. Williams, J.G., Gratzer, W.B. (1971) J. Chromat.7, 121-125. 26. Huisman, J.G., Stapel, S., and Muijsers, A.O.M. (m78) Febs Letters 85, 198-202. 27. Boulter, D., Ellis, R.J., and Yarwood, A. (1972) Biol. Rev. g,

28. 29. 30.

1131175;

Davies, J.W., and Kaesberg, P. (1973) J. Virol. 12, Bottomley, W., Higgings, T.J.V., and Whitfeld, P.R. Febs Letters 63, 120-124. Seckbach, J. n972) J. Ultrastruct. Res. 2, 65-76.

1131

1434-1441. (1976)