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The nematode worm C. elegans, with its transparent body, is an excellent vehicle for studying developmental gene expression during embryogenesis and throughout its short life. Expression data from in-situ hybridization, immunolocalization and reporter constructs have been put into the ACeDB database, which is used to store and disseminate most types of C. elegans data, and is also widely used for genome-sequencing projects. In the database, the gene-expression patterns are linked to genes, sequences, cells, organs and the developmental stage in which expression occurs. An accessory program 'Angler' can be used to browse sectional Nomarski images of the worm embryo during early development, and to relate these images to overlaid cell lineage data and 3-D schematic views of cell positions.Copyright 1997 Academic Press Limited Copyright 1997Academic Press Limited

In this review of protein synthesis, we have described a system for translation of mRNA using extracts of A. nidulans. This system is useful for characterizing mutants suspected to have defects in protein synthesis and for assessing the toxicity of various antibiotics and their effects on misreading the genetic code. The well developed genetical system of A. nidulans has enabled us to map at least 27 new genes whose mutation disturbs the level of translational accuracy. These mutants could be used to identify new components of translation or new roles for established components. The abundant fidelity mutations themselves could be used to elucidate the mechanism for maintaining the accuracy of protein synthesis. The large number of mutations in the control of fidelity indicate that mutations in other parts of the translation system could be easily obtained. This would be particularly important for initiation where many factors are thought to be needed and yet their exact roles are unknown. A. nidulans appears to have normal eukaryotic ribosomes and translation factors that can be used to study the mechanism of protein synthesis, its regulation, and the maintenance of its high fidelity. If highly purified factors were used, requirements for hitherto undiscovered factors could be seen. Since A. nidulans has typical eukaryotic responses to inhibitors of translation, it could be used to study new inhibitors, their mode of action, and their potency. Among the fungi, A. nidulans could be a worthy competitor to S. cerevisiae in the field of protein synthesis, particularly because so many translation genes have been identified. The system awaits further exploitation.

Mutants of Aspergillus nidulans resistant to the aminoglycoside antibiotics paromomycin and hygromycin B have been isolated and their growth characteristics are described here. Most paromomycin mutants were cross-resistant to hygromycin and geneticin. All the hygromycin-resistant mutants were slightly cross-resistant to geneticin. Out of the 15 mutants tested 14 had drug-resistant ribosomes in vitro and all 12 of those investigated further had reduced levels of translational misreading. Five new loci have been found--parA on linkage group I, hygA on III, hygB on IV, hygC on V, hygD on VI and parB on VIII. This increases, to at least 12, the number of translational fidelity loci in A. nidulans.

Antisuppressor mutations were isolated in a strain containing the omnipotent suppressor suaC109. The antisuppressors reduce the activity of translational suppressors in vivo and counteract most aspects of the pleiotropic phenotype associated with the suaC and the suaA suppressor mutations. Using an homologous system for cell-free translation, we have measured translational accuracy in two antisuppressor strains with the genotype suaC109 and either the asuB11 or the asuD14 antisuppressor mutation. Ribosomes from antisuppressor mutants have higher levels of translation accuracy than those from the suppressor strain (suaC109, asu+). Mistranslation levels depended solely on the source of the sucrose-cleaned ribosomes. However, the increased accuracy associated with sucrose-cleaned ribosomes from antisuppressor strains can be nullified by salt-washing, suggesting that the component responsible can be washed off.

A third type of translational fidelity mutation has been induced in Aspergillus nidulans. The new mutation enhances growth, in suppressing conditions, of a strain containing suppressor suaC109 and antisuppressor asuD14 and is called aloB8 for its allosuppressor activity. Compared with the progenitor strain (asuD14, suaC109), ribosomes from the new mutant (aloB8, asuD14, suaC109) increase misincorporation of leucine in a poly(U)-dependent homologous cell-free assay. The misreading level is maintained by ribosomes after washing with 500 mM KCl and suggests an alteration in a ribosomal component or a tightly bound factor. Ribosomes from an aloB8, asu+, sua+ strain also misread to a higher level than those of the control strain despite the fact that, in vivo, this mutation alone has no known suppressor activity. Mg2+ ions have been used in vivo for the first time to classify translation mutants.

Three genotypically suppressible alleles, a1X4, alcA125, and niaD500, are phenotypically suppressed by aminoglycoside antibiotics. Unsuppressible alleles at these loci are unaffected as are known missense mutations at the yA and gdhA loci. This is consistent with the premise that the suppressible mutations are nonsense and that this highly-allele-specific phenotypic suppression can be used to distinguish nonsense from missense mutations of Aspergillus nidulans. Paromomycin and tobramycin are recommended for screening unknown mutations.

A series of strains, doubly mutant at conidiation loci, have been made. The phenotypes of these strains reflected the epistasy of earlier blocking mutants over later ones and confirmed the order of gene sequence predicted from the phenotypes of single mutants. Oligosporogenous mutants gave complex interactions, especially between brl and med mutants. These results indicated that (i) gene action overlapped in time, (ii) several parts of the conidial apparatus were interchangeable and (iii) nuclei leaving the vesicle were not irreversibly programmed. Structures produced by mutants were reminiscent of the conidial apparatus of other Aspergillus species and of related genera.

Strains of Aspergillus nidulans containing informational suppressors were grown on medium containing antibiotics known to affect protein synthesis at the ribosomal level. These strains reacted in the anticipated manner: presumed ribosomal suppressors suaA101, suaA105, suaC109 and sua-115 were sensitive or even hypersensitive to aminoglycoside antibiotics, whereas presumed tRNA-like suppressors suaB111, suaD103 and D108 were only slightly sensitive or wild-type in response. Hygromycin and paromomycin were the most useful antibiotics. All the antibiotics reduced the colony radial growth rate, Kr, increased the lag phase and produced wrinkled morphology. Hygromycin was the most toxic. Resistant sectors were produced on paromomycin and hygromycin. The selective action of 'misreading' antibiotics on suaA and suaC strains is further evidence that these are ribosomal suppressors, whereas suaB and suaD may code for altered tRNA molecules. The results imply that hygromycin or paromomycin could be used for isolating ribosomal suppressors.

18 suppressors of alcR125 have been selected in Aspergillus nidulans. They have been located in genes as follows: 12 in suaA, 1 in suaB and 5 in suaC. Suppressors have been examined to see whether their phenotype is diagnostic for their genotype. Several new traits are described: conidial viability, cycloheximide resistance, fertility, suppression of niaD500, niaD501 and fwA1. These tests, added to those already in use, provide a battery of tests suitable for assigning suppressor mutations to physiological type (tRNA or ribosomal), and in one case to a specific gene since only suaA mutations suppressed fwA1. A very broad range of phenotypes was associated with suppressors such that every mutation had a unique phenotype. This indicates that the ribosomal suppressor mutations are in genes which code directly for ribosomal proteins, rather than genes which code for modifying enzymes.

Ammonium ions are selectively toxic to strains containing mutations in the suaC gene which can mutate to a suppressor phenotype. This phenotype is associated with increased ribosomal misreading in vitro (Zamir and Martinelli 1987) and altered ribosomal proteins (Harvey and Martinelli 1983). Such ammonium-sensitivity is a feature of both strong and weak suppressor alleles, and segregates with suppressor ability in crosses. Suppressor mutations in the suaB and suaD genes are not affected, nor are those in suaA, another ribosomal suppressor gene. Thus, the ammonium-effect is locus specific. Mutations which act as anti-suppressors (asu-) of suppressor suaC109 also partially reverse the ammonium ion sensitivity associated with this mutation. This effect is in line with their restoration of other aspects of the pleiotropic phenotype to normal. The cations, lithium and rubidium, mimick the effects of ammonium ions. Only ribosomes from suaC strains are sensitive to the presence of NH+4 ions in vitro.