AMINO ACID REQUIREMENTS OF THE FREE-LIVING NEMATODE CAENORHABDITIS BRIGGSAE

BY

J. R. VANFLETEREN Instituut voor Dierkunde, Laboratoria voor Morfologie en Systematiek, RijksuniversiteitGent, Belgium

Washed yeast ribosomes promote growth and reproduction of C. briggsae, even when supple- mented to the basal medium at dosages too low to provide the organisms with sufficient amounts of essential amino acids. Hence, a re-investigation of the amino acid requirements of C. briggsae by single and multiple omission of amino acids from the basal medium revealed unambiguously that arginine, histidine, lysine, tryptophan, phenylalanine, methionine, threonine, leucine, isoleucine and valine are not synthetized at levels to permit reproduction; they are called essential amino acids. The requirement for arginine and isoleucinehowever appears to be less clear-cut. On the contrary, evidence is presented that alanine, asparagine, cysteine, glutamate, glutamine, glycine, proline, serine and tryosine can be synthetized at adequate levels; they are called non- essential amino acids. In addition it was shown that multiple omission of the non-essential amino acids is not deleterious. This is believed to be an important step towards the development of a minimum essential medium (MEM) for growth and reproduction of C. briggsae.

Sustained growth of the free-living nematode Caenorhabditis brigg.rae can be obtained on a chemically defined medium, supplemented with adequate levels of a proteinaceous growth factor. The most satisfactory, chemically defined medium hitherto reported (Buecher, Hansen & Yarwood, 1966), has been called C. brigg.iae Maintenance Medium (CbMM) and is now commercially available. CbMM is an extremely rich medium, being composed of 53 components, all present at high concentrations. The level of many components is probably unnecessarily high (Sayre, Hansen & Yarwood, 1963); other components might be non-essential and could be omitted. Precise nutritional studies are however difficult to perform in the presence of unidentified material such as the growth factor (Sayre et al., 1963). Thus Dougherty, Hansen, Nicholas, Mollet & Yarwood (1959) could only give preliminary evidence that the following amino acids are essential: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. More insight in the amino acid metabolism was gained by adding radioactive amino acid precursors to the medium and subsequent determination of the labeled amino acids by chromatography and radioautography. The biosynthesis of 16 amino acids could be demonstrated with this technique: aspartic acid, glutamic acid, alanine, proline, glycine, cysteine and cystine (as cysteic acid) and serine (Nicholas, Dougherty, Hansen, Holm-Hansen & Moses, 1960; Rothstein & Tom- linson 1961, 1962); arginine, threonine, tyrosine, valine, leucine, isoleucine, 94 histidine and lysine (Rothstein & Tomlinson, 1961, 1962). The biosynthesis of tryptophan, methionine and phenylalanine has not been reported so far, but cannot be excluded since these amino acids are easily lost or destroyed under the conditions of isolation (Nicholas et al., 1960; Rothstein & Tomlinson, 1962). The use of data, obtained from radioactive labelling seems to be very limited for getting more insight in the amino acid requirements of C. briggJae. As already outlined by Nicholas et al. ( 1 960) and Rothstein & Tomlinson (1961, 1962), the level of synthesis adequate to permit growth and reproduction cannot be deter- mined by this method. Recently, new and highly active growth promoting substances have been reported (Buecher, Hansen & Gottfried, 1969, 1970; Lower & Buecher, 1970). With one of them (yeast ribosomes), we have obtained excellent growth, when supplemented to CbMM at considerably reduced levels (expressed as protein content) as compared with the amounts of protein formerly administered (Dougherty et al., 1959). In our opinion, a re-examination of the nutritional requirements of C. briggsae in the light of this feature could be more rewarding. The elucidation of the amino acid requirements of C. brigg.rae might be an important step towards the development of a minimum essential medium. The availability of such a medium is indispensable for the isolation and identification of nutritional mutants.

MATERIALSAND METHODS

Highly purified ribosomes were prepared from bakers' yeast cake as described by Lower & Buecher (1970) (preparation of yeast extract) and Buecher et al., (1969) (isolation and purification of ribosomes from yeast extract) with slight modification. Briefly, yeast cake (550 g) was mixed with equal volumes of glass beads 0.2 mm in diameter and Tris-Mg buffer (5 X 10-`' M Tris; 5 X 10- M = MgC12; PH 7.6) and homogenized in a Waring Blendor (one gallon capacity) with cooling periods between grindings until approximatively 80% of the cells were disrupted. The homogenate was filtered through cheese cloth, centrifuged for 20 minutes at 10,000 X g and the supernatant again for 30 minutes at 30,000 X g. The ribosomes were subsequently sedimented at 105,000 X g for 3 hours and resuspended in Tris-Mg buffer. The suspension was treated addi- tionally with deoxyribonuclease for further purposes and washed twice as described by Buecher et al. (1969). The final suspension was filtered through Millipore filters of diminishing porosity and sterilized by passing through a sterile Millipore PH (0.30 membrane. The protein content was 4.9 mg/ml as determined by both the biuret (Robson, 1968) and Lowry (Lowry et al., 1951) methods; the DNA content 2.75 ttg/ml), estimated by the method of Burton (1956) can be neglected. Aliquots of 1 and 2 ml were transferred into sterile culture tubes and stored at -18° C until needed, because it has been observed that repeated freezing and thawing causes a loss of activity; in addition a general contamination is prevented.