Chemosensory control of surface antigen switching in the nematode Caenorhabditis elegans.

Nematodes change their surface compositions in response to environmental signals, which may allow them to survive attacks from microbial pathogens or host immune systems. In the free-living species Caenorhabditis elegans, wild-type worms are induced to display an L1 (first larval stage) surface epitope at later larval stages when grown on an extract of spent culture medium (Inducible Larval Display or ILD). Before this study, it was not known whether ILD was regulated by the well-characterized, neurologically based chemical senses of C. elegans, which mediate other behavioural and developmental responses to environmental signals such as chemotaxis and formation of the facultatively arrested dauer larva stage. We show here that ILD requires the activities of three genes that are essential for the function of the C. elegans chemosensory neurons. ILD was abolished in chemotaxis-defective che-3, osm-3 and tax-4 mutants. In contrast, chemotaxis-defective mutants altered in a different gene, srf-6, show constitutive display of the L1 epitope on all four larval stages. The ILD-defective che-3, osm-3 and tax-4 mutations blocked the constitutive larval display of an srf-6 mutant. Combining srf-6 and certain dauer-constitutive mutations in double mutants enhanced constitutive dauer formation, consistent with the idea that srf-6 acts in parallel with specific components of the dauer formation pathway. These results taken together are consistent with the hypothesis that ILD is triggered by environmental signals detected by the nematode's chemosensory neurons.

Environmental induction and genetic control of surface antigen switching in the nematode Caenorhabditis elegans.

Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two process can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly.

Caenorhabditis elegans as a model for parasitic nematodes: a focus on the cuticle.

The phylum Nematoda consists of over half a million species of worms that inhabit astoundingly diverse environments. Nematodes can live as obligatory parasites of plants and animals, or alternate a parasitic with a free-living life style. The fact that the vast majority of species are strictly free living often surprises parasitology students, for obviously the highest research priorities in this field have involved parasites of medical, veterinary and agricultural importance. Here Samuel Politz and Mario Philipp contend that some basic questions concerning the biology of the parasite cuticle can be investigated more easily and in greater depth in the free-living nematode Caenorhabditis elegans than in the parasites themselves.

Altered expression of an L1-specific, O-linked cuticle surface glycoprotein in mutants of the nematode Caenorhabditis elegans.

Mouse mAb M38 was used in indirect immunofluorescence experiments to detect a stage-specific antigen on the surface of the first larval stage (L1) of the free-living nematode Caenorhabditis elegans, and to detect alterations in the apparent expression of this antigen in two distinct classes of C. elegans mutants. In previously described srf-2 and srf-3 mutants (Politz S. M., M. T. Philipp, M. Estevez, P.J. O'Brien, and K. J. Chin. 1990. Proc. Natl. Acad. Sci. USA. 87:2901-2905), the antigen is not detected on the surface of any stage. Conversely, in srf-(yj43) and other similar mutants, the antigen is expressed on the surface of the first through the fourth (L4) larval stages. To understand the molecular basis of these alterations, the antigen was characterized in gel immunoblotting experiments. After SDS-PAGE separation and transfer to nitrocellulose, M38 detected a protein antigen in extracts of wild-type L1 populations. The antigen was sensitive to digestion by Pronase and O-glycanase (endo-alpha-N-acetylgalactosaminidase), suggesting that it is an O-linked glycoprotein. This antigen was not detected in corresponding extracts of wild-type L4s or srf-2 or srf-3 L1s, but was detected in extracts of srf-(yj43) L4s. The antigen-defective phenotype of srf-3 was epistatic to the heterochronic mutant phenotype of srf-(yj43) in immunofluorescence tests of the srf-3 srf-(yj43) double mutant, suggesting that srf-(yj43) causes incorrect regulation of a pathway of antigen formation that requires wild-type srf-3 activity.

Genes that can be mutated to unmask hidden antigenic determinants in the cuticle of the nematode Caenorhabditis elegans.

Rabbit antisera directed against a mixture of proteins solubilized from the wild-type adult Caenorhabditis elegans cuticle were used to isolate mutants, induced by ethyl methanesulfonate treatment, that exhibit alterations in surface antigenicity by immunofluorescence. Genetic mapping and complementation data for four such mutations define two genes, srf-2(I) and srf-3(IV). The mutant phenotypes observed by immunofluorescence appear to result from unmasking of antigenic determinants that are normally hidden in the wild-type cuticle. In support of this hypothesis, surface radioiodination experiments indicate that components labeled on the wild-type surface are missing or less readily labeled on the surface of srf-2 and srf-3 mutants.

Genetic analysis of adult-specific surface antigenic differences between varieties of the nematode Caenorhabditis elegans.

We have studied developmental stage-specificity and genetic specification of surface antigens in the nematode Caenorhabditis elegans. Rabbit antisera directed against the adult C. elegans cuticle were used in conjunction with antiserum adsorption experiments to obtain antibody reagents with specificity for the adult surface. Adult-specific antibodies were used to identify several varietal strains of C. elegans that display antigen-negative phenotypes as adults. Genetic mapping results using the surface antigen phenotype as a marker indicated that a single gene (designated srf-1) or cluster of genes on linkage group II determines the adult surface antigen phenotype.

Small Collagenous Proteins Present during the Molt in Caenorhabditis elegans.

Immunoblotting experiments using antibodies directed against the large collagenous cuticle proteins of Caenorhabditis elegans revealed a class of small collagenous proteins (CP) of apparent molecular weight 38,000-52,000 present during the L4 to adult molt. These CP are smaller than most vertebrate collagens characterized to date and share many characteristics with the small collagenous products translated in vitro from RNA isolated at this molt. C. elegans collagen genes, collagen-coding mRNA, and collagenous in vitro products that have been characterized are also small. Detection of small CP in vivo in C. elegans thus lends further support to the hypothesis that such small collagenous proteins are the primary gene product precursors to the larger collagenous proteins isolated from the C. elegans cuticle.

Ribonucleic acid-protein cross-linking in Escherichia coli ribosomes: (4-azidophenyl)glyoxal, a novel heterobifunctional reagent.

We have used the heterobifunctional reagent (4-azidophenyl)glyoxal (APG) to cross-link RNA to protein in Escherichia coli 30S ribosomal subunits. Synthesis and characterization of the reagent are described. Like other dicarbonyl reagents (e.g., kethoxal), APG reacts specifically with guanosine among the four ribonucleosides. The azido group in APG can be photolyzed with UV light (lambda greater than 300 nm), yielding an unstable nitrene which is potentially reactive with many groups in proteins and nucleic acids. Conditions for APG modification of guanylic acid residues in 30S subunits are described; photolysis of bound APG results in cross-linking of approximately 5% of the total 30S proteins to 16S RNA. A specific subset of the 30S proteins is cross-linked to 16S RNA by APG.

Magnesium-dependent interaction of 30S ribosomal subunits with antibodies to N6, N6-dimethyladenosine.

The modified nucleoside N6, N6-dimethyladenosine occurs in Escherichia coli 16S ribosomal RNA only in two successive positions near its 3' end. Antibodies directed against dimethyladenosine were induced with a nucleoside-albumin conjugate. As measured by second antibody precipitation of immune complexes, antidimethyladenosine antibodies bound 30S ribosomal subunits, ribosomal core particles, and ribosomal RNA which contain dimethyladenosine but showed little cross-reactivity with RNA or ribosomal subunits from a kasugamycin-resistant mutant which lacks dimethyladenosine. Antibody binding to ribosomal subunits was strongly influenced by the concentration of magnesium ion in the reaction medium and by the prior treatment of the subunits. Functionally active 30S subunits showed a striking binding optimum at 2-4 mM Mg2+; this optimum disappeared if the subunits were inactivated by dialysis against low concentrations of magnesium ion. Instead, the inactivated subunits showed a gradual increase in antibody binding as the magnesium ion concentration was raised to 20 mM; binding of 16S ribosomal RNA or subribosomal core particles from 30S subunits gave qualitatively similar curves, with no evidence of a low [Mg2+] optimum. The stability of antibody-subunit complexes was also found to depend upon subunit conformation and magnesium ion concentration; the half-life of an inactivated subunit-antibody complex (15 mM Mg2+) averaged 130 min, while active subunit-antibody complexes (3 mM Mg2+) had an average half-life of 70 min. More of the immune complexes with inactivated subunits were found to survive sucrose gradient sedimentation (relative to active subunits), and the concentration of subunits needed to halve antibody binding of [3H]-N6, N6-dimethyladenosine was lower with inactivated subunits. The results suggest that the antibody binding optimum seen with active subunits at 2-4 mM Mg2+ represents a dynamic aspect of the three-dimensional ribosomal subunit structure; a site near the 3' end of the RNA is involved, and both the availability of the modified nucleoside to an antibody probe and the stability of the resulting complexes are involved.

Ribosome structure: localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex.

Antibodies to the minor nucleoside N6,N6-dimethyladenosine have been used to map a unique location of the nucleoside in the small subunit of the Escherichia coli ribosome. Antibodies were induced in rabbits by a nucleoside-bovine albumin conjugate and shown to be highly specific for the dimethyladenosine hapten. The antibodies were shown to interact with 30S ribosomal subunits from strain PR7, but not with subunits from its mutant strain TPR201, which is resistant to kasugamycin and lacks the two successive residues of dimethyladenosine normally found near the 3'-end of E. coli 16S ribosomal RNA. Electron micrographs of strain PR7 subunits, crosslinked by single IgG molecules, show a single binding site on the surface of the ribosome. This binding site is consistent with observations relating the 3'-end of the ribosomal RNA, binding of initiation factor IF-3 and messenger RNA, and mapping of specific ribosomal proteins.