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Biochimica et Biophysica Acta.562 (1979) 547--551 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 91481

REGULAR, RODLIKE, MACROMOLECULAR STRUCTURES IN ARTEMIA SALINA

P A U L N I E U W E N H U Y S E N a, FRAN~OISE RIJSHEUVELS a, H E R M A N SLEGERS a and WIM JACOB b

aDepartment of CellBiology and bDepartment of Medicine, Universityof Antwerp, Universiteitsplein 1, B 2610 Wilrijk (Belgium) (Received January 2nd, 1979)

Key words: Nucleoprotein complex; Ribosome; Electron microscopy; (Artemia salina)

Summary Artemia salina of different origins and in different developmental stages contains regular, rodlike, macromolecular structures. The particles were visualized by electron microscopy after negative staining. They can be purified from the postmitochondrial supernatant by sucrose gradient centrifugation in the presence of EDTA. Their size distribution has been measured from electron micrographs. The particles are probably nucleoprotein complexes.

A recent report [1] and bibliography containing more than 1000 references [2], reflect the interest in various aspects of the brine shrimp Artemia salina, such as its morphology and biochemistry, and its use in aquaculture. The fertilized eggs of A. salina develop in the female ovisac until free-swimming nauplii are liberated (ovoviviparity) or the development is stopped in the gastrula stage as a consequence of desiccation, the gastrulae are encysted and shed into the environment (ovoparity); after rehydration these cysts can further develop to nauplii [3, 4]. The occurrence of these two developmental pathways and of the period of dormancy in which no endogeneous protein synthesis takes place makes A. salina very useful for the study of stored, masked messenger ribonucleoproteins. During electron microscopy of ribosomes and messenger ribonucleoproteins of A. salina, we have detected regular rodlike, macromolecular structures. These have been examined further and a procedure for their purification has been developed [ 5]. Electron micrographs of the structures are shown in Fig. 1. The particles were first observed in partially purified ribosome fractions, obtained by isopycnic centrifugation of postmitochondrial supernatant of the cryptobiotic

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Fig. 1. E l e c t r o n m i c r o g r a p h s of t h e r o d l i k e s t r u c t u r e s p r e s e n t in A . salina. S a m p l e s w e r e n e g a t i v e l y s t a i n e d w i t h a s o l u t i o n of 2% u r a n y l a c e t a t e in distilled w a t e r a n d v i s u a l i z e d w i t h a J E O L - I O O B transm i s s i o n e l e c t r o n m i c r o s c o p e o p e r a t i n g at 8 0 kV. S t a i n i n g w i t h p h o s p h o t u n g s t i c acid y i e l d e d less c o n t r a s t . F i x a t i o n w i t h f o r m a l d e h y d e p r i o r t o staining c a u s e d n o r e m a r k a b l e d i f f e r e n c e . T h e s t r u c t u r e of t o b a c c o m o s a i c v i r u s w a s m u c h less c l e a r w h e n v i s u a l i z e d in t h e s a m e c o n d i t i o n s . Scale bars: 1 0 0 A.

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embryos of A. salina in a high-density sucrose gradient, using the Beckman R 60 rotor [6]. In a further study, they were found predominantly in the sucrose gradient fractions of densities 1.27 to 1.30 g/cm 3. These fractions contain also ribonucleoproteins [7]. It should be noted that the buoyant density of ribosomes and other (ribo)nucleoproteins is much lower in sucrose than in the more conventional CsC1 density gradient centrifugation [6, 7]. The particles were also observed in resuspended crude ribosome pellets, obtained after a high-speed centrifugation (4°C, 50 min, 250 000 X gay) of the postmitochondrial supernatant of the cryptobiotic embryos. This is consistent with the fact that they were found to sediment slightly ahead of the 80-S ribosomes in sucrose gradients. The structures were also directly detected in the postmitochondrial supernatant of nauplii and in the hemolymph of adult shrimps. The particles were still observed after treatment with high concentrations of CsC1 and with the Mg-chelating agent EDTA. Ribosomes however are dissociated by EDTA into slowly sedimentJng forms of their subparticles. We therefore purified the rodlike structures by zonal centrifugation of concentrated postmitochondrial supernatant in 15--50% sucrose gradients containing 25 mM EDTA to eliminate the complete ribosomes (Fig. 2). Following this procedure the particles were found to be present in the cryptobiotic embryos of different origins such as San Francisco Bay {U.S.A.), Utah Lake (U.S.A.), and Madras (India}. The dimensions of the particles (Fig. 3) have been estimated from electron micrographs. Their apparent diameter is about 22 nm. Their length is heterogeneous (with a maximal value around 300 rim), since the structures are often broken during the isolation and preparation for electron microscopy (Fig. 1), as observed also with rodlike virus particles. The high buoyant density of the particles in sucrose suggests that they are nucleoprotein complexes, as noted above. Their composition was further examined by incubation with proteinase K, DNAase and RNAase. Undegraded particles from incubation mixtures and controls were centrifuged on films I0-~267mi 5

(

0 bottom

I 10 Fraction number

I 20

top

Fig. 2. I s o l a t i o n o f t h e m a c r o m o l e c u l a r s t r u c t u r e s f r o m p o s t m i t o c h o n d r i a i s u p e r n a t a n t o f c r y p t o b i o t i c e m b r y o s o f A. salina. 1 m l , c o n t a i n i n g 9 0 A 260 o f p o s t m i t o c h o n d r i a l s u p e r n a t a n t in p h o s p h a t e buffer [ 6 ] w a s l o a d e d o n a l i n e a r 1 5 - - 5 0 % sucrose g r a d i e n t in p h o s p h a t e buffer c o n t a i n i n g 2 5 m M E D T A . C e n t r l f u s a t i o n w a s a t 4 ° C in t h e B e c k m a n , SW 2 7 r o t o r a t 2 4 5 0 0 r e v . / m i n ( 8 0 0 0 0 × g a y ) f o r 8 h. T h e structures w e r e l o c a l i z e d i n the i n d i c a t e d fractions. ( T h e a b s o r b a n c e p e a k s arc due t o r i b o s o m a l p a r t i c l e s w h i c h s e d i m e n t s l o w l y because o f the E D T A . )

550 0

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1C

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20

30 DIAMETER(rim)

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b

1

i

I

//A

N

8

"'6 ~A Z

..

0

i

i

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100

200

300

400 Length (nm)

Fig. 3. Diameter (a) and length (b) distribution (determination by electron microscopy) of regular, rodlike structures isolated by zonal centrifugatlon from the postmitoehondrial supernatant of the cryptobiotic embryos of A. salina.

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supported by copper grids [8]. Comparison in the electron microscope showed a pronounced proteinase and a less pronounced DNAase sensitivity; the particles were not degraded by RNAase. So these results indicate that the structures are nucleoproteins. The difference in sensitivity for proteinase and DNAase may even be explained by a partial protection of the nucleic acid by a protein coat. The resemblance of the structures with rodlike virus particles is striking. References 1

Jaspers, E. (1977) Fundamental and Applied Research on the Brine Shrimp A r t e m i a salina (L.), European Marlculture Society, Bredene, Belgium 2 Sorgeloos, P. (1976) A Bibliography on the Brine Shrimp A r t e m i a salina (L.), European Mariculture Society, Bredene, Belgium 3 Finamore, F.J. and Clegg, J.S. (1969) in The Cell Cycle (Padilla, G.M. et al., eds.), Academic Press, New York 4 Hentschel, C.C. and Tata, J.R. (1976) Trends Biochem. SeL 1, 97--100 5 Nieuwenhuysen, P., Rijsheuvels, F., Jacob, W.A. and Slegers, H. (1978) 12th FEBS Meeting, Dresden, Abstract No. 2220 6 Nieuwenhuysen, P. and Slegere, H. (1978) Anal. Biochem. 89, 472--480 7 Slegers, H. and Kondo, M. (1977) Nucl. Acids Res. 4, 625--639 8 MttUer, G. (1969) Arch. Gesamte Virus Forschung 27, 339--342

Regular, rodlike, macromolecular structures in Artemia salina.

547 Biochimica et Biophysica Acta.562 (1979) 547--551 © Elsevier/North-Holland Biomedical Press BBA Report BBA 91481 REGULAR, RODLIKE, MACROMOLECUL...
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