Worm Breeder's Gazette 13(2): 83 (February 1, 1994)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
sqt-1 and rol-6 encode collagens that we can now confidently call cuticle collagens EMS and in vitro o n-mutagenesis of sqt-1 and rol-6 revealed conserved amino acids in the amino and carboxyl non-Gly-X-Y domains that are important for normal function (see Worm Meeting Abstract, 1993). Mutations in carboxyl cysteines generate left roller (LRol) animals ( sc13 and sc113 ).Loss of the strong positive charge at position 1 or 4 of Homology Block A (HBA R(1), V(2), R(3), R(4), Q(5)) causes a dominant right roller (RRol), recessive dumpy (Dpy) phenotype ( e1350 and sc1 ).The conserved pattern of arginines of HBA suggests that HBA may be the cleavage site of a subtilisin-like endoproteinase.
In order to examine mutations at the biochemical level, we have generated antipeptide antisera against SQT-1 .On Western blots affinity purified antisera react to cuticle extracts from N2 but not sqt-1 null mutant animals, demonstrating the specificity of the antiserum. In N2 cuticle extracts two major bands at 70 kDa and 120 kDa and some higher molecular weight material are seen to react. We believe that the 70 kDa molecule represents a dimer of two collagen chains based on the size of SQT-1 after the predicted cleavage at HBA (25 kDa) and the fact that collagens migrate 40% slower than expected on SDS-PAGE. The two collagen chains in the dimer and the larger multimers, are presumably di/tri-tyrosine cross-linked. The bulk of cuticle collagens migrate at 90-98kDa (trimer), indicating that the cross-linking of Sqt-1 differs from the majority of collagens.
Cuticle extracts from sqt-1 mutants show different SQT-1 patterns. The dominant RRol HBA mutants show very weak reactivity, indicating that most of these mutant SQT-1 molecules do not reach the cuticle presumably due to lack of cleavage at HBA. The LRol mutants show reduced levels of the very high molecular weight material and an increased amount of an approximately dimer size (68 kDa) molecule that runs slightly faster than in wild-type, indicating that the cysteine replacement mutations affect the formation of non-reducible crosslinks.
To detect SQT-1 before it has been extensively crosslinked during cuticle assembly, we have examined extracts from molting animals 100 molting worms (mostly L4 /Ad)were picked directly into sample buffer (containing SDS and BME) and boiled before loading. Compared to cuticle extracts, the extracts from N2 molting worms show increased intensity of the 70 kDa dimer band and a weak band at 36 kDa, expected to be a SQT-1 monomer. Extracts of molting LRol animals allow the faster migrating (68 kDa) dimer band of reduced intensity and a very heavy 36 kDa (monomer) band. The SQT-1 monomer (36 kDa), dimers of wild-type (70 kDa) and LRol (68 kDa) are all disulfide bonded into the cuticle since they are only solubilized in the presence of BME. The accumulation of the 36 kDa SQT-1 monomers in LRol mutants indicates that mutation of the carboxyl cysteine inhibits dimerization. Since the cysteine is immediately preceded by a conserved tyrosine, it appears that the loss of disulfide bonding inhibits the neighboring tyrosine from participating in cross-linking.
We have also made antipeptide antisera against the ROL-6 cuticle collagen The preliminary results show that in N2 cuticle extracts ROL-6 has two major bands that are similar, but not identical in size, to the SQT-1 bands. This result indicates that SQT-1 and ROL-6 are not crosslinked together by nonreducible bonds