Induction of multiple proteases during the early larval development of Artemia salina

Extracts from dormant and developing embryos of Artemia salina contain almost undetectable levels of protease activity. However, after hatching of the nauplii, there is a high increase in the activity of the extracts on different proteolytic substrates. The activities correspond to four different enzymes, A, B, C, and D, that have a sequential timing of induction during the early larval development. The enzymes show a different elution pattern after chromatography on DEAE-cellulose and a different sensitivity to protease inhibitors. Enzyme A is strongly inhibited by phenylmethylsulfonyl fluoride, while the other enzymes are mostly insensitive. Enzymes A, B, and C are sensitive at low concentrations of the soybean trypsin inhibitor. The temporal induction of these enzymes is an example of the expression, at the biochemical level, of the developmental program of Artemia salina.     

The role of guanine nucleotides in the interaction between aminoacyl-tRNA and elongation factor 1 of Artemia salina.

The low-molecular-weight form of elongation factor 1 (EF-1L) of the cysts of the brine shrimp Artemia salina and [3H]phenylalanyl-tRNA are able to form a stable complex which can be isolated on a Sephacryl S200 column. The formation of this complex is inhibited by increasing concentrations of magnesium acetate and KCl. Furthermore, the formation of this complex is independent of the presence of guanine nucleotides. Complex formation between EF-1L and phenylalanyl-tRNA appears to be specific, since acylation of the tRNA is a necessity for this interaction. Although EF-1L alone binds GDP somewhat more strongly than GTP, the complex between EF-1L and phenylalanyl-tRNA binds GTP exclusively. Our results support the idea that complex formation between EF-1L and aminoacyl-tRNA precedes the enzymatic binding of aminoacyl-tRNA to the 80-S ribosome. Subsequently to this binding, release of EF-1L from the ribosome occurs.     

Membrane associated cytoplasmic mRNA in Artemia salina; functional and physical changes during development.

The physical and functional properties of the mRNA population from developing embryos of the brine shrimp Artemia salina were characterized. About 20% of the total poly(A)-rich mRNA in these embryos appears to be specifically associated with the membrane fraction throughout early development, and physically differs markedly from the free cytoplasmic mRNA. The membrane-associated mRNA fraction consists of two well-defined populations of molecular weight of 5.2x10(5) and 3.6x10(5), whose relative amount changes during the various stages of embryo development. The size of the poly(A) tail at the 3'-end of the mRNA molecules, as estimated by processive phosphorolysis, was found to consist of 180 and 210 adenosine residues for the two respective mRNA species. The in vitro translation products of the membrane-bound mRNA molecules are apparently similar to those of the free mRNA molecules.     

Physical properties of Artemia salina ribosomes.

Eukaryotic ribosomes were isolated from the cryptobiotic embryos and from the further-developed free-swimming nauplii of the brine shrimp Artemia salina. Analytical boundary sedimentation and photon correlation spectroscopy yielded, respectively, the standard sedimentation and diffusion coefficients at infinite dilution, s degrees 20,w = 81 +/- 1 S and D degrees 20,w = (1.41 +/- 0.02) x 10(-7) cm2/s, for the unfixed and formaldehyde-fixed ribosomes from different developmental stages and for ribosomes attached to a messenger RNA fragment. Also, the density increment was determined, from which the partial specific volume was derived (0.63 +/- 0.01 cm3/g). Combination of the different measured parameters gives accurate values for the molecular weight (3.8 +/- 0.1) x 106 and for size and solvation parameters. These results are compared with their counterparts for the smaller ribosomes from the prokaryote Escherichia coli.     

Characterization of the extracellular haemoglobins of Artemia salina.

The following factors were measured for extracellular haemoglobins of Artemia salina: a minimal molecular weight of globin chain per haem group (based on the iron and haem contents), the absorption coefficients, the absorption spectra of various derivatives and the amino acid compositions. These were compared with those of the haemoglobins of other invertebrates. Three Artemia haemoglobins (I, II and III) had similar molecular structures, constructed from two-globin subunits of 122000-130000mol.wt. Since the minimal mol.wt. was determined to be 18000, this suggests that one globin subunit was bound by seven haem groups, and hence one haemoglobin molecule (240000-260000mol.wt.) should contain 14 haem groups. A successful identification of this high-molecular-weight subunit required first the denaturation of haemoglobin in 1% sodium dodecyl sulphate before sodium dodecyl sulphate gel electrophoresis. Denaturation by prolonged incubation (12-36 h) at room temperature in the presence of 0.1% sodium dodecyl sulphate [Bowen, Moise, Waring & Poon (1976) Comp. Biochem. Physiol. B55, 99-103] was accompanied by extensive proteolysis, resulting in low recovery of the stainable protein and heterogeneous gel patterns. Regardless of which electrophoretic system was used, the high-molecular-weight subunit was always present provided that 1% sodium dodecyl sulphate was present during denaturation. These results contrast with those obtained by Bowen et al. (1976). However, preferential cleavage of the globin subunit (alpha) seemed to occur in vitro when standard conditions were used, producing two specific fragments having mol.wts. of 80000 (beta) and 50000 (gamma).     

Molecular cloning and expression of four actin isoforms during Artemia development.

Four cDNA clones coding for different Artemia actin isoforms have been isolated. Three of the clones contain the complete coding sequences while the fourth one lacks 145 bases, coding for the 49 amino terminal amino acids of the protein. The amino acid sequences predicted for the four actin isoforms identified are highly homologous to insect actins as well as to vertebrate cytoplasmic actins. The four identified cDNA clones code for mRNAs of 5.2, 1.9, 1.6 and 1.8 kb, respectively, whose expression is regulated during development. Three of the actin mRNAs are present in cryptobiotic embryos while the other is not. The steady-state levels of all four mRNAs increase during development to reach maximal levels by 10-15 hours of development and decrease thereafter. The total number of actin genes encoded in the Artemia genome has been estimated as 8 to 10 by Southern analysis of total DNA.     

Antennular sensilla of the brine shrimp, Artemia salina.

1. Scanning electron microscopy was used to characterize the external morphology of setae found on the antennules of adults and nauplii of the brine shrimp, Artemia salina (L.). The permeability of the antennular setae was studied by means of Slifer's crystal violet method. 2. Each antennule of an adult brine shrimp possessed a terminal cluster of sensory setae. Within a cluster there were two morphologically distinct kinds of sensilla, here designated type 1 and type 2. Three type 1 sensilla were observed on every antennule examined. The number of type 2 sensilla per antennule was usually four or five. 3. Type 1 sensilla of adults were 43 to 80 micrometer long and simple in external morphology. They were widest at the base, decreased in diameter gradually, and terminated as a finely tapered tip. No pores were resolved by scanning electron microscopy. 4. Type 2 sensilla of adults were shorter (shaft length, 12 to 23 micrometer) and displayed a single pore at the tip (average pore diameter, 0.4 micrometer). In thin section they were seen to possess a distinctive articular specialization of the cuticle at the base of the seta. 5. Dye penetration experiments indicated that type 2 sensilla were permeable to aqueous crystal violet, whereas type 1 sensilla were not. 6. The antennular setae of nauplii resembled type 1 sensilla in general shape, in being impermeable to crystal violet, and in lacking a terminal pore and basal articular specialization. Moreover, a total of three setae was normally present on each naupliar antennule, and the same number of type 1 sensilla was found on each adult antennule examined. If the three naupliar setae represent a developmental stage in the formation of three adult sensilla, available observations suggest that the larval setae are developmentally related to type 1, rather than to type 2 adult sensilla.     

The genetics of Artemia salina. VII. Reproductive isolation.

Fifteen of 20 gonochoristic Artemia populations are crossfertile with diploid San Francisco shrimps, producing fertile F1 and viable F2 progeny. Partial sex linkage of white eye was observed and frequency of crossing over between the white and sex loci did not exceed the range of values observed in San Francisco shrimps. Possible mechanisms for wide dispersal of this diploid genotype are discussed. Five populations are reproductively isolated from San Francisco shrimps: Mono Lake, Hidalgo, Lake Urmia, San Bartolomeo, and Tunisia. The last two are inter-fertile.     

Structure of extracellular hemoglobin from the brine shrimp Artemia salina.

1. Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and preparative gel electrophoresis in non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with Mr 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.     

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina.

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.