[26] The use of Artemia salina for developmental studies: Preparation of embryos, tRNA, ribosomes and initiation factor 2

298

I N I T I A T I O N OF P R O T E I N S Y N T H E S I S

[26]

[ 2 6 ] T h e U s e o f A r t e m i a s a l i n a for D e v e l o p m e n t a l Studies: Preparation of Embryos, tRNA, Ribosomes and Initiation Factor 2 By A. H. WARNER, T. H. MACRAE, and A. J. WAHBA

The importance of the brine shrimp, Artemia salina, in the study of developmental biochemistry has increased in recent years.'-'° This increase is due, partially, to the availability of Artemia as highly stable, dormant, early gastrula (cysts), which remain metabolically inactive until hydrated in the presence of oxygen.".'" Upon hydration at 30 °, morphogenesis proceeds in a relatively synchronous manner, without cell division, for 8 hr, eventually resulting in a swimming larva by 24 hr (Fig. 1). Such characteristics, in concert with low levels of protease and nuclease activities in cysts and newly hatched larvae,13"4 offer obvious advantages for the study of embryogenesis. The main disadvantage of encysted Artemia embryos as a developmental model is their impermeability to virtually all important radiolabeled precursors of nucleic acids and proteins. Two bisexual, diploid ''~,'6 populations of Artemia cysts, from the San Francisco Bay area (California) and the Great Salt Lake (Utah) are commercially available. The Utah embryos produce a greater quantity of NH3 '7 and contain higher quantities of ATPase, TM dipeptidase, acid protease, 'r and 80 S ribosomes 1'~than do California embryos. The acid-soluble nucleoF. J. Finamore and J. S. Clegg, in "The Cell Cycle: Gene-Enzyme Interactions" (G. M. "Padilla, I. L. Cameron, and G. L. Whitson, eds.), p. 249. Academic Press, N e w York, 1969. 2 j. C. B a g s h a w , F. J. Finamore, and G. D. Novelli, Develop. Biol. 23, 23 (1970). '~ H. C. Birndorf, J. D ' A l e s s i o , and J. C. Bagshaw, Develop. Biol. 45, 34 (1975). 4 W. Filipowicz, J. M. Sierra, C. Nombela, S. Ochoa, W. C. Merrick, and W. F. Anderson, Proc. Natl. Acad. Sci. U.S.A. 73, 44 (1976). S. M u t h u k r i s h n a n , W. Filipowicz, J. M. Sierra, G. W. Both, A. J. Shatkin, and S. Ochoa, J. Biol. Chem. 250, 9336 (1975). 6 H. Grosfeld and U. Z. Littauer, Eur. J. Biochem. 70, 589(1976). r D. K. McClean and A. H. Warner, Develop. Biol. 24, 88 (1971). 8 F. L. Huang and A. H. Warner, Arch. Biochem. Biophys. 163, 716 (1974). 9 A. H. Warner, V. Shridhar, and F. J. F i n a m o r e , Can. J. Biochem. 55, 965 (1977). ,0 j. S. Clegg and A. L. Golub, Develop. Biol. 19, 178 (1969). 11 R. D. Ewing and J. S. Clegg, Comp. Biochem. Physiol. 3 1 , 2 9 7 (1969). ,2 D. M. Stocco, P. C. Beers, and A. H. Warner, Develop. Biol. 27, 479 (1972). ~:~A. H. Warner, unpublished observations, 1977. ,4 p. Nagainis and A. H. Warner, unpublished observations, 1977. 1.~T. Iwasaki, Jpn. J. Genet. 44, 105 (1969). in S. T. Bowen, Biol. Bull. 126, 333 (1964). ,r L. Bellini and D. M. de Vincentiis, Exp. Cell Res. 2 1 , 2 3 9 (1960). ,s S. Russo-Caia and L. Bellini, Rend. Ist. Sci. Univ. Camerino 1, 136 (1960). METHODS IN ENZYMOLOGY, VOL. LX

Copyright© 1979by AcademicPress. Inc. All rights of reproductionin any form reserved. ISBN 0-12-181960-4

[26]

Artemia salina FOR DEVELOPMENTALSTUDIES

299

.,p

FIG. I. Artemia salina embryosat various stagesof development.C, cyst; P, prenauplius larva; N, nauplius larva. tide pools appear, however, to be the same in both populations. ,3 Thus, Utah and CaliforniaArtemia populations have distinctly different biomasses but appear to be similar biotypes. Materials

Buffer A: 50 mMTris-HCl, pH7.4; 100mM KC1; 10mM Mgacetate; 1% sodium dodecyl sulfate (SDS) Buffer B: 50 mM Tris.HCl, pH 7.4;. 5 mM KCI; 10 mM acetate; 1 mM dithiothreitol (DTT) or 10 mM 2-mercaptoethanol. Buffer C: 50 mM Tris .HCI, pH 7.4; 50 mM KC1; 10 mM Mg acetate; 25 % glycerol; 1 mM DTT Buffer D: 50 mM Tris. HC1, pH 7.4; 100 mM KCl; 10 mM Mg acetate; 1 mM DTT Buffer E: 50 mM Tris.HCl, pH 7.4; 500 mM NH4C1; 5 mM Mg acetate; 1 mM DTT Buffer F: 50 mM Tris-HCl, pH 7.4; 700 mM NH4C1; 10 mM Mg acetate; 1 mM DTT Buffer G: 50 mM Tris .HCI, pH 7.4; 200 mM KCI: 10 mM Mg acetate: I mM DTT Buffer H: 20 mM Tris.HCl, pH 7.4; 100 mM NH4CI; 1 mM Mg acetate Buffer I: 20 mM Tris- HCI, pH 7.4; 100 mM NH4CI; 5 mM Mg acetate Buffer J: 50 mM Tris. HCI, pH 7.4; 100 mM KCI; 0.2 mM Mg acetate; 10% glycerol; 10 mM ME Buffer K: 50 mM Tris.HCl, pH 7.4; 0.2 mM Mg acetate; 10% glycerol; 10 mM 2-mercaptoethanol Buffer L: 50 mM Tris. HCI, pH 7.4; 50 mM KCi; 0.2 mM Mg acetate; 10% glycerol; 10 mM 2-mercaptoethanol

300

I N I T I A T I O N OF P R O T E I N S Y N T H E S I S

[26]

Sepharose 6B-100, from Sigma Chemical Co. Ultrogei AcA 44, from LKB Sephadex G-25 (fine), from Pharmacia DEAE-cellulose (DE-52 and DE-11), from Whatman Inc. [:~sS]Methionine (specific activity 437-566 Ci/mmol), from New England Nuclear Corp. Yeast tRNA (unfractionated), from Boehringer Mannheim ApUpG, from Miles Laboratories GTP, from P.L. Biochemicals, Inc. Ammonium sulfate (ultrapure) and sucrose (ultrapure), from Schwarz/Mann Artemia salina cysts: Utah biotype, from Longlife Aquarium Products, Division of Sternco Industries, Inc., Harrison, New Jersey 07029 or St. Thomas, Ontario, Canada. California biotype, from Metaframe Corporation (San Francisco Bay Brand), 893 Enterprise Dr., Newark, California 94560.

Preparation of Reagents Hatch medium: 422 mM NaCI; 9.4 mM KC1; 25.4 mM MgSO4; 22.7 mM MgCI.,; 1.4 mM CaCI2; 0.5 mM N a H C Q ; 1000 units of penicillin G (Na) per milliliter; and 100/xg of streptomycin sulfate per milliliter TM Sea water is hatch medium with 1321 mM NaC1 Antiformin stock solution: 7.8 g NaOH and 3.2 g NazCO3 dissolved in 100 ml of Clorox or 5.7% NaOCI. Before use 7 ml of stock solution are diluted to 100 ml with incubation medium. The stock solution should be stored at 4 ° in the dark. [35S]Met-tRNA: [35S]Met-tRNA (11,000-13,000 cpm/pmol) is prepared by charging unfractionated yeast tRNA with [33S]methionine in the presence of Escherichia coli synthetase. "°,21 AminoacyltRNA preparations are passed through a Sephadex G-25 column (18 cm × 0.9 cm) to remove contaminating nucleotides. Preparation of Artemia Salina Embryos

Hydration, Washing, and Sterilization of Cysts Commercially available brine shrimp cysts are usually heavily contaminated with sand, dry matter, microorganisms, and nonviable cysts. 19 A. H. W a r n e r and D. K. McClean, Develop. Biol. 18, 278 (1968). z0 U. L. R a j B h a n d a r y and H. P. G h o s h , J. Biol. Chem. 244, l l04 (1969). 21 N. K. Gupta, N. K. Chatterjee, K. K. Bose, SI Bhaduri, and A. C h u n g , J . Mol. Biol. 54, 145 (1970).

[26]

Artemia salina FOR DEVELOPMENTAL STUDIES

301

Except for the latter, all contaminants can be easily removed and washed cysts are suitable for the isolation of proteins and nucleic acids. For washing, cysts are hydrated for 3 hr with occasional stirring at 4 ° in 3-5 volumes of 0.5 M NaCI. After hydration, the cyst suspension is diluted 3-fold with cold distilled H.,O and cysts are allowed to settle for 20 min. The Utah cysts are more dense than those from California, and they sediment in a few minutes. The California cysts are composed of two subpopulations, one containing a heavy shell and the other a light shell."" Thus, some of the California cysts will float in the wash/hydration medium and the remainder will sediment. A few cysts will also remain suspended. We have found that both the " f l o a t e r s " and " s i n k e r s " of California cysts yield about the same number of nauplii. Floating cysts, "'floaters," are decanted and the remaining cysts, " s i n k e r s , " are washed 4 - 6 times with several volumes of cold H~O. The cysts are then collected by filtration either on a coarse frittedglass filter or on 4 - 6 layers of cheesecloth in a Bfichner funnel. Care is taken to avoid sand and debris at the bottom of the container. The cysts are washed with 1-2 liters of distilled H._,O and rinsed with buffer if desired. Washed cysts are used immediately or stored overnight in an ice bath. For isotope incorporation with cysts or nauplii, cysts must be sterilized or radioactivity is incorporated into the bacterial flora attached to the cyst chorion. Methods used to disinfect Artemia cysts include the use of 0.1% Merthiolate, '-'a,'4 !% Merthiolate and 0.1% NaOCI, ea 5% NaOC1, ''' 7% antiformin,'.2~ and commercial Clorox (5.7% NaOCI) diluted 1: I with HeO. To obtain sterile cysts we use an antiformin or a Clorox treatment. All solutions should be filter-sterilized, and glassware heat-sterilized. For antiformin sterilization, dry cysts are suspended in 4-8 volumes (25-50 g/200 ml) of ice-cold 0.5 M NaCI and mixed well. Fifteen milliliters of antiformin solution are added, and the cyst suspension is incubated, with occasional mixing, for 30 rain. Clorox sterilization requires a 20-rain incubation of cysts in 5 volumes of Clorox diluted 1:! with cold H.,O. This procedure removes the chorion of the cysts 27along with associated bacteria and spores and renders the cysts aseptic. Prolonged treatment of the cysts with antiformin should be avoided to prevent clumping of the cysts and eventual reduction in yield of nauplii upon incubation. After Sterilization, the suspension is diluted 2-4 fold with H20, and the cysts are allowed to partition between the top and bottom of the beaker. Cysts are washed as described above and the sinkers are collected by e2 j. Dutr!z::, Arch. Zool. Exp. Gen. 99, 1 (1960). '-':' L. Provasoli and K. Shiraishi, Biol. Bull. 117, 347 (1959). 24 j. S. Clegg, J. Exp. Biol. 4 1 , 8 7 9 (1964). '-':' J. Renart and J. Sebastian, Cell Dill: 5, 97 (1976). 2, y . H. Nakanish[, T. lwasaki, T. Okigaki, and H. Kato, Armor. Zool. Jpn. 35, 223 (1962). 27 j. E. Morris and B. A. Afzelius, J. Ultrastruct. Res. 20, 244 (1967).

302

I N I T I A T I O N OF P R O T E I N SYNTHESIS

[26]

filtration, washed with H20 and suspended in 0.5 M NaCI for about 1 hr. Hydrated cysts are again collected by filtration, washed with 5% urea to remove traces of NaOC1, and then with water and buffer. Should the bacteria-free cysts contain fungi, the addition of 0.1% benzalkonium chloride 28 to the 0.5 M NaCI solutions removes these contaminants. The yield of fully hydrated and sterile cysts is variable and dependent on the level of debris and floating cysts in the population. However, 100 g of dry dormant cysts from California or Utah yield approximately 100 or 200 g, respectively, of fully hydrated cysts.

Incubation Procedures Brine shrimp develop aerobically in saline medium at temperatures up to 40°. 23'~9 Maximum hatching, with Utah cysts, is obtained with a saline medium that resembles the sea water in the embryos' natural environment?° This medium, fortified with penicillin and streptomycin sulfate to suppress bacterial growth, is called hatch medium. After hatching, embryos are maintained in sea water. Small-Scale Incubation Procedure. To obtain 100,000-200,000 developing embryos, i-2 g of fully hydrated, sterile cysts are placed in a 100 × 80 cm glass storage dish. Seventy-five milliliters of Millipore-filtered hatch medium are added, and the dish is covered and incubated at 30° on a platform shaker at 75-80 excursions/min. At the desired developmental stage (Fig. 2) the embryos are collected on a fritted-giass funnel (30 ml, coarse) and washed with water and then with the medium that will be used for homogenization. Large-Scale Incubation Procedures. To 50 g of fully hydrated, sterile cysts, in a Fernbach culture flask, 750 ml of sterile hatch medium are added. The flask is covered and incubated at 30° on a platform shaker at 80-100 strokes/rain. Developed embryos are collected on a fritted-glass funnel and washed as above. Large batches of developing embryos are required for the preparation of polypeptide chain initiation factors. Three hundred grams (wet weight) of washed, sterile cysts are added to 3 liters of hatch medium in a 6-liter Erlenmeyer flask. The flasks are incubated at 30° with shaking and vigorous aeration. At the appropriate developmental stage, the embryos are harvested and washed. Development is somewhat slower and less efficient than in less crowded conditions, and this procedure is not recommended if hatched embryos are desired. z~ F. M. Rottman, personal communication. z9 E. Bonne and L. G. M. Bass-Becking, J. Gen. Physiol. 14, 753 (1931). 3o G. E. Hutchinson, " A Treatise on Limnology," p. 269. Wiley, N e w York, 1957.

[26]

Artemia salina FOR DEVELOPMENTALSTUDIES I00

303

"Colifornio

80

Nouplii

6O

4o o

6"

20 ,

J

E I00 E laJ 80

/ ~ X X.x ~( , / , , ,"'X'~.---,-...K

Prenouplii j

~

J

,

,

v,

Utoh

60 Nouplii

40 20

} I

I

6

~ 12

~ 18 Hours

24 at

--i :50

=

Prenauplii

56

42

i

,

i ~1

48

30 °

FIG. 2. Developmental profile of Artemia salina embryos from California and Utah salterns.

Collection of Prenauplii During the first 8 hr of development at 30°, following the termination of dormancy by hydration and aeration, Artemia embryos undergo morphogenesis while encased in their chitinous shell. During the next 3-4 hr the prenauplius larvae, contained in a hatching membrane, begin to emerge from the shell, and most embryos remain attached to the shell by the hatching membran& 6 (Fig. 1). Utah prenauplii, attached to their shell, can be readily separated from undeveloped cysts by transferring the embryos to a large finger bowl or culture dish (8.5 × 25 cm) and swirling the embryos gently for 5-10 sec. Undeveloped cysts remain on the bottom of the dish while the prenauplius larvae collect in a ring near the periphery of the dish and are collected with the large end of a pipette. This procedure, which is most effective with 50-75 g of embryos, is repeated and can result in populations containing over 90% prenauplii. California cysts remain suspended in hatch medium and tend to hamper collection of prenauplii by the method just described. Thus, to collect prenauplii from California brine shrimp, emerged embryos attached to cyst shells are collected, by suction, from the surface of culture flasks left stationary for 10-15 min after incubation. The membrane-enclosed prenauplii and empty cyst shells are separated by the suction. The shells float

304

INITIATION OF PROTEIN SYNTHESIS

[26]

to the surface of the collection flask and are removed by suction, whereas the prenauplii settle to the bottom of the flask and are collected on a coarse filter.

Collection of Nauplii The phototactic response of newly hatched swimming nauplii permits their large-scale harvest. After hatching has begun (Fig. 2) nonviable and undeveloped embryos are allowed to settle in a beaker. Nauplii and floating cysts are decanted into a separatory funnel, the top three-fourths of which is maintained in darkness. An incandescent light is directed at the base of the funnel, and gathering nauplii are collected by opening the stopcock. This procedure is repeated until the nauplii are free of unwanted cysts and embryos. Aseptically collected nauplii may be maintained, without feeding, for 2-3 days in sea water, before significant bacterial or fungal growth occurs.

Determination of Cyst Viability The viability of commercially availableArtemia cysts varies considerably and should be determined before initiating biochemical or developmental studies. To determine viability, 1.0 ml of cyst suspension from a small-scale incubation is diluted, shortly after the start of incubation, with 3-4 ml of 0.5 M NaCl. One milliliter is removed from the thoroughly mixed dilution and transferred quickly along a 2.5 X 56 cm strip of Whatman No. I filter paper lined at I-cm intervals. The cysts on the paper surface are counted with the aid of a dissecting microscope and mechanical counter. Four to five hundred cysts per paper strip can be easily counted with an accuracy of + 7% when performed in triplicate. The remainder of the cysts are incubated at 30° for at least 24 hr, after which I-ml samples are removed, diluted, and spread on filter paper strips. The prenauplii and nauplii are counted. From these data, the percentage of embryos that have emerged (prenauplii) or hatched (nauplii) (Fig. 1) and in turn, cyst viability, can be determined. In general, Utah cysts develop more synchronously than California cysts, whereas the latter yield more developing embryos (Fig. 2). Hatched and unhatched Utah embryos have similar nucleotide pools, and the reason for their lack of emergence and hatching is unknown, r_,

Breakage of Cysts and Mixed Populations of Embryos Fully hydrated encysted embryos, compared to desiccated cysts, offer little resistance to breakage. Techniques used to fractureArtemia embryos

[26]

A r t e m i a salina FOR DEVELOPMENTAL STUDIES

305

include stirring at high speed with a magnetic stirrer,~','~z grinding with a Dounce-type tissue grinder ~,33or a mortar and pestle~,7,9'34 and homogenization with the Duall or Ten Broeck type ground glass homogenizers. ,4,2~,The electric torsion mortar grinder, Model MG-2, manufactured by the Torsion Balance C o m p a n y (Clifton, New Jersey) efficiently disrupts large batches of desiccated or hydrated cysts. Its use is detailed in the following sections. Subcellular fractions are obtained from nauplii by placing 8-10 g, wet weight, of nauplii in a Dounce homogenizer tube, adding 4 volumes of homogenizing medium, and gently homogenizing until the homogenate has a fine consistency. Large quantities of nauplii ( 100-200 g) can be efficiently homogenized with a Waring blender. ~ Nauplii should be processed quickly at 0° to avoid artifacts produced by trypsin-like proteases, which are abundant in naupfii older than 24 h r ? 5 P r e p a r a t i o n of A . s a l i n a Cyst t R N A a n d R i b o s o m e s P r e p a r a t i o n o f A . salina C y s t t R N A

Fifty grams ofA. salina cysts, in 10-g batches, are ground dry at room temperature for 5 min with a motorized mortar and pestle (Model MG-20). The cyst powder is stirred for 1 hr at 4 ° with 400 ml of buffer A. The buffer must be mixed well before addition to the ground cysts as the SDS does not go into solution. After stirring; the suspension is centrifuged at 7000 rpm for 30 min (Sorvall, GSA rotor) and the supernatant collected by passing through cheesecloth and glass wool. An equal volume of 90% phenol, equilibrated with buffer A without SDS, is added to the supernatant and stirred for 30 rain. The aqueous phase is collected by aspiration, and reextracted with 0.5 volume of 90% phenol as just described. The aqueous phases are combined; the tRNA is precipitated by the addition of 3 volumes of cold ( - 2 0 °) ethanol and left overnight at - 2 0 °. The ethanol-insoluble precipitate is collected by centrifugation at 9000 rpm for 30 rain and dried at room temperature. Drying time here, and in subsequent steps, is shortened by passing nitrogen over the pellet. Fifty milliliters of 1 M NaCI are added to the dry pellet, and the mixture is incubated at 4 ° for 3 hr with occasional shaking. The suspension is centrifuged at 9000 rpm for 20 min, the supernatant is saved, and the sediment :~' A. H. Warner, J. G. Puodziukas, and F. J. Finamore, Exp. Cell Res. 70, 365 (1972). :~2C. G. Vallejo, M. A. G. Sillero, and A. Sillero, Biochim. Biophys. Acta 358, 117 (1974). :~'~A. Golub and J. S. Clegg, Develop. Biol. 17, 644 (1968). :~4M. Zasloff and S. Ochoa, Proc. Natl. Acad. Sci. U.S.A. 68, 3059 (1971). :~ P. Nagainis, M.Sc. Thesis, University of Windsor, Windsor, Ontario, 1976.

306

[26]

INITIATION OF PROTEIN SYNTHESIS

is reextracted with 25 ml of I M NaCI. The supernatants are pooled and stored overnight in an ice bath. The pooled supernatants, containing about 2000 A.,~o units, are diluted 5-fold with cold distilled water and applied to a DE-11 cellulose column (3.5 × 23 cm) previously washed with 500 ml of 1.0M NaCI and 500 ml of 0.20M NaCI. After sample application the column is washed, at a flow rate of 3-4 ml/min, with 500 ml of 0.2 M NaCI and 800 ml of 0.35 M NaC1. The tRNA, collected in 15-ml samples, is eluted with 1 M NaCI. The tRNA is concentrated by overnight precipitation, at --20 °, with 3 volumes of cold ethanol. The ethanol-insoluble precipitate is collected by centrifugation at 9000 rpm for 20 min, dried, and redissolved in 3.5 mi of 1.8M Tris .HCI, pH 8.2. The tRNA is deacylated by incubation at 37° for 1 hr. The preparation, which contains about 300 A260 units, is diluted 10-fold with water, and the tRNA is precipitated overnight with 3 volumes of ethanol at - 2 0 °. The precipitate from the preceding step is dissolved in 2.5 ml of 50 mM KCI and applied to an Ultrogel AcA 44 column (2.5 X 35 cm) previously equilibrated with 50 mM KCi at room temperature. The column is eluted with 50 mM KCI, and 5-ml samples are collected at a flow rate of I ml/min (Fig. 3). The fractions from peak 3 are combined, and the tRNA is precipitated with 3 volumes of ethanol and left overnight at --20 °. The precipitate is collected by centrifugation at 10,000 rpm for 20 min, dried, and dissolved in 1 ml of water. The yield is approximately 100 A.,60 units of tRNA.

Peak 3

6

0

4

o

~ 3 Peak 2

g 2 Peak I

- i

20

-

-i

60

~

_

I00

140 Effluent

180

220

260

300

340

volume ( m l )

FIG. 3. Ultrogel AcA 44 chromatography ofArternia salina cyst tRNA. Tubes from peak 3 contain the tRNA.

[26]

Artemia salina FOR DEVELOPMENTALSTUDIES

307

Preparation of Unwashed Ribosomes Seventy-gram batches of hydrated, washed cysts, rinsed with buffer B, are ground for 5 min at 4° in a motorized mortar and pestle. Each 70 g of ground cysts is resuspended in 20-25 ml of buffer B, and all batches are combined. The disrupted cysts are stirred for 15 min and centrifuged at 8000 rpm for 20 min. The 8000 rpm supernatant is centrifuged at 17,000 rpm for 30 min, and the resulting supernatant, except for that in the bottom fifth of the centrifuge tube, is removed with a 50-ml syringe. The ribosomes are collected from the 17,000 rpm supernatant by centrifugation at 46,000 rpm for 2 hr (Spinco, 60 Ti rotor), rinsed with buffer C, and resuspended in the same buffer to give a final A.,60of about 500 units/ml. The ribosomes pellets can be dispersed by gentle homogenization or by stirring at 4° for 3-4 hr with a magnetic stirrer. After the suspension is centrifuged at 10,000 rpm for 15 min (Beckman, JA-20 rotor), the absorbancy is determined and the ribosomes are stored at - 7 0 °. Six hundred grams of dry cysts yield 10,000-12,000 A.~;0 units of ribosomes.

Chromatography of Ribosomes on Sepharose 6B The activity of cyst ribosomes in translation of artificial mRNA [poly(U)] is improved by passage of the ribosomes over a column of Sepharose 6B. s Solid KCI is added, to a final concentration of 0.1 M, to 1000A,,60units of unwashed cyst ribosomes in buffer C. The ribosome suspension is centrifuged at 10,000 rpm for 15 min, the pellet is discarded, and the supernatant is applied to a Sepharose 6B column (2.5 × 88 cm) equilibrated with buffer D at 4°. The ribosomes are eluted with the same buffer at a flow rate of about 50 ml/hr (Fig. 4). Peak 1 is cloudy and pigmented, consisting of glycogen and some ribosomes. Peak 2 fractions are clear and contain ribosomes which are collected by centrifugation at 46,000 rpm for 2 hr (Spinco, 60 Ti rotor). Ribosome activity is improved by centrifuging ribosomes through a cushion of buffer containing 25% glycerol. The ribosome pellets are resuspended in buffer C, dispersed by gentle homogenization and stored at --70 °. Approximately 350 A2,0 units of ribosomes are recovered from the column.

Salt-Washed Ribosomes Unwashed ribosome pellets collected as previously described are rinsed with buffer B, resuspended in buffer E, and slowly stirred overnight at 4 °. The ribosomes are collected by centrifugation at 46,000 rpm for 2 hr (Spinco, 60 Ti rotor), rinsed with buffer C and resuspended in the same

308

[26]

INITIATION OF PROTEIN SYNTHESIS

,6f

Peak

15

14

13 12 II I0 9

ID

g

8

7! 6 5 4 3 2 I

50

I00

150 200 250 E f f l u e n t volume (ml)

300

350

FIG. 4. Sepharose 6B chromatography ofArtemia salina cyst ribosomes. Peak 1 contains glycogen and some ribosomes whereas peak 2 consists of ribosomes.

buffer. Ribosome pellets may be dispersed by gentle homogenization or by stirring for 3-4 hr at 4°. The ribosome suspension is centrifuged at 10,000 rpm for 15 min before the absorbancy is determined. Six hundred grams of dry cysts yield approximately 10,000 A260 units of 0.5 M NH4CI washed ribosomes. The ribosomes are stored at --70 °.

Cyst 40 S Ribosomal Subunits Cyst ribosomes, which have been prepared by washing with 0.5 M NH4CI, are rinsed with buffer F and then resuspended in this buffer. The pellets are dispersed by stirring with a glass rod, followed by incubation in an ice bath for 2-6 hr. The ribosome suspension is centrifuged at 10,000 rpm for 15 min. The absorbancy of the resulting supernatant is adjusted to 200 A26 o units/ml by dilution with buffer F. Two milliliters of the ribosome suspension are applied to 36 ml, 15 to 30% continuous sucrose gradients in buffer F, and centrifuged at 24,000 rpm for 14 hr (Spinco, SW 27 rotor). The gradients are fractionated by using an Auto Densi-Flow 11 C (Buchler Instruments) and a Gilford 250 spec-

[26]

Artemia salina FOR DEVELOPMENTALSTUDIES

309

trophotometer. The pooled 40 S subunit preparation, collected from the ascending side of the 40 S peak, is diluted with an equal volume of buffer G, and the subunits are collected by centrifugation at 40,000 rpm for 10 hr (Spinco, 60 Ti rotor). The pellets are resuspended in 0.5 ml of buffer G, yielding approximately 40A ,,6ounits of 40 S subunits. When stored at --70 °, the ribosomal subunits remain active, for at least 4 months, in chain mltmtlon assays. Subunit purity is analyzed by applying 1A._,~0unit in 0.1 ml of buffer G to analytical 5-ml 15 to 30% continuous sucrose gradients in buffer G. The gradients are centrifuged at 50,000 rpm for 80 min (Spinco, SW 65 rotor) and when analyzed with the Auto Densi-Flow I1 C yield one peak. Preparation of Initiation Factor 2 (eIF-2) from Artemia Cysts Determination of elF-2 Activity Assay of Ternary (Met-tRNAfGTP.elF-2) Complex Formation. This reaction measures the amount of [:~S]Met-tRNA bound to the chain initiation factor (eIF-2) in the presence of GTP. The reaction mixture contains, in 50 p~l, 20 mM Tris .HCI, pH 7.4, 100 mM NH4C1 and/or KCI, 1 mM Mg acetate, 0.16 mM GTP, 2 pmol of ['~zS]Met-tRNA, 1 mM DTT, and partially purified factor ranging from 0 to 100/.~g per assay mixture. After incubation for 10 min at 30°, the reaction mixture is diluted with 3-4 ml of cold buffer H, filtered through a nitrocellulose filter (0.45 p~m), which is washed, dried and counted. One unit of eIF-2 activity is defined as 1 pmol of ['~S]MettRNA bound to the filter under standard conditions. Specific activity, determined from the linear portion of the curve obtained by plotting pmol of [:~'~S]Met-tRNA bound to the filter vs amount of protein per reaction mixture, is expressed as units of activity per milligram of protein. Protein is quantitated by the method of Lowry et al. 36 with bovine serum albumin as standard. Initiation Complex Formation. The assay measures the binding of ['~sS]Met-tRNA to 40 S ribosomal subunits. Assay mixtures contain, in a volume of 0.1 ml, 20 mM Tris. HCI, pH 7.4, 100 mM KCI, 3.7 mM Mg acetate, 4.7 pmol of [:~sS]Met-tRNA, 0.85 A260 unit of 40 S ribosomal subunits, 84 p~gof cyst eIF-2 from DEAE-cellulose, 1 mM DTT, and, when present, 0.16 mM GTP and 0.1 A.,60 unit of AUG. After incubation for 10 min at 30°, reaction mixtures are layered on 5.0 ml, 15 to 30% linear sucrose gradients in buffer G, and centrifuged at 50,000 rpm for 2 hr (Spinco, SW 65 rotor). After centrifugation, fractions are collected with the aid of the Auto 3~O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall,J. Biol. Chem. 193, 265 (1951).

310

[26]

INITIATION OF PROTEIN SYNTHESIS B

40S

~_71

/ \

' (140

0.30

/~Com.,e,e - 0.20 o 5

2

o.lo

/

<

2

4

6

8

I0 12 Froction

number

FI6. 5. Formation of 40 S initiation complex. Unless otherwise indicated, 84 p.g of Met-tRNA binding factor from DEAE-cellulose was added to each reaction. Sedimentation was from left to right. (A) Complete reaction mixture E]---D: no factor added O---O. (B) No AUG added I - - - I ; no GTP added 0 - - - 0 .

Densi-Flow 11 C, diluted with buffer I and filtered through nitrocellulose filters (0.45/~m). The filters are washed three times with buffer I, dried, and counted. As shown in Fig. 5, formation of an initiation complex with Artemia cyst 40 S ribosome subunits is dependent on eIF-2, AUG, and GTP.

Preparation of Artemia Cyst eIF-2 Step 1. Preparation ofO.5 M NH4CI Ribosomal Wash. Unwashed cyst ribosomes from 300 g wet weight of cysts are suspended in 75 ml of buffer E and slowly stirred overnight at 4°. The ribosomes are removed by centrifugation at 46,000 rpm for 2 hr (Spinco, 60 Ti rotor). Step 2. Ammonium Sulfate Fractionation. Powdered (NH4)2SO4 (14.6 g) is slowly added to 70 ml of high-salt ribosomal wash, stirred for 20 min, and centrifuged at 10,000 rpm for 15 min (Beckman, JA-20 rotor). An additional 12.7 g of (NH4)2SO 4 is added to the supernatant, the mixture is stirred for 20 min, and the precipitate is collected by centrifugation. This precipitate (35 to 60% (NH4)2SO4 fraction) is dissolved in 6.0 ml of buffer J and dialyzed extensively against 1 liter of the same buffer. The precipitate that forms during dialysis is removed by centrifugation at 10,000 rpm for 15 min. Step 3. DEAE-Cellulose Chromatography. The above supernatant is diluted with an equal volume of buffer K and incubated in an ice bath for 30 min. The resulting precipitate is removed by centrifugation at 10,000 rpm for 15 rain, and the supernatant, containing about 115 mg of protein, is applied to a DE-52 cellulose column (31.5 X 1.2 cm) previously equili-

[27]

311

PROKARYOTIC RIBOSOME B I N D I N G SITES

KCI, mM ~ j

I' 1.1-

~J

N

I

~ o.9

4~ g03 z<°~ v

0.7 0.5 0.3 o.t

~

~

.,j

~X, '

10

20

30

40

50

-

6(3

~

7'0

1 ,~

8'0

"

~

9'0

-

100

140

Fraction number

FIG. 6. DEAE-cellulose chromatography of 35 to 60% a m m o n i u m sulfate fraction from Artemia salina cyst ribosomal wash. The gradient was started at fraction 16. Aliquots of each fraction (40 t~l) were assayed for GTP-dependent [3'~S]Met-tRNA binding as described. Each reaction mixture contained 4 pmol of [3~S]Met-tRNA in a total v o l u m e of 100/zl.

brated with buffer L. The column is washed with buffer L until no further protein is eluted. A linear gradient of 50 to 350 mM KC1 in a total volume of 200 ml of buffer K is then applied. The flow rate is 15 ml/hr, and fraction size is 2 ml (Fig. 6). The specific activity at this stage of purification ranges from 7 to 15 units per milligram of protein. Purification is approximately 20-fold relative to elF-2 activity in the ribosomal wash.

[27] P r o k a r y o t i c R i b o s o m e

By

Binding Sites

JOAN ARGETSINGER STEITZ

Takanami, Yan, and Jukes 1 first demonstrated that ribosomes could protect associated segments of natural messenger RNA (mRNA) from ribonuclease digestion in 1965. A few years later, three other groups adapted their basic procedure to isolate protein synthesis initiator regions from the genomes of the small RNA-containing coliphages. '-4 Sequence analysis of these messenger fragments showed that, in in vitro reactions M. Takanami, Y. Yan, and T. H. Jukes, J. Mol. Biol. 12, 761 (1965). J. A. Steitz, Nature (London) 224, 957 (1969). :3 j. Hindley and D. H. Staples, Nature (London) 224, 964 (1969). 4 S. L. Gupta, J. Chen, L. Schaefer, P. Lengyel, and S. M. Weissman, Biochem. Biophys. Res. Commun. 39, 883 (1970).

METHODS IN ENZYMOLOGY, VOL. LX

Copyright© 1979by Academic Press, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-181960-4