Eddy SR (1994) Worm Breeder's Gazette "Three Families of Reverse Transcriptase Elements in C. elegans"

Eddy SR

[Worm Breeder's Gazette, 1994]

Three families of reverse transcriptase elements in C. elegans Sean Eddy, MRC-LMB, Hills Road, Cambridge CB2 2QH, UK

Manser J et al. (1985) Worm Breeder's Gazette "Autosomal Extragenic Suppressors Of her-1(n695) V"

Wood WB, Manser J, Trent C

[Worm Breeder's Gazette, 1985]

We are studying five extragenic mutations that suppress the Tra phenotype of her-1(n695) V (see preceding abstract): ct27 III (gamma- ray induced), n1091 III and ct49 III (allelic, EMS-induced; ct49 was isolated by Sean Burgess), and n1092 V and n1102 V (allelic, EMS- induced). ct27 III has a semi-dominant dpy phenotype in XX animals; XO animals are less severely Dpy and are abnormal males. Both ct27; n695 and ct27/+;n695 XX animals exhibit strong suppression of the n695 Tra phenotype. Because ct27 results in a phenotype somewhat similar to that caused by mutations in the chauvinist dpy genes (dpy's 21, 26, 27, 28) it may suppress n695 with an effect on X-chromosome expression. n1091 III has no apparent effect on XX or XO animals by itself, while ct49 III XX and XO animals are Dpy h we have not eliminated the possibility that this phenotype results from a second closely linked mutation). n1091;n695 XX animals are well suppressed; n1091/+;n695 XX animals are not. n1092 V and n1102 V map near unc-76 (about 5% from her-1). Neither appears to have any effect on XX or XO animals by itself. For n1092 and n1102, both n695 sup and n695 sup/n695 + XX animals show strong suppression. n695n1O91 XO, n695n1O92 XO, and n695n11O2 XO animals are all normal males (that is, they are apparently identical to n695 XO animals). This suggests that the EMS-induced suppressor mutations on III and V affect a process that is active in XX, but not XO, animals. For example, the genes identified by these mutations could be involved in turning off her-1 expression in XX animals.

Ouazana R (1980) Worm Breeder's Gazette "isolation and biochemical analysis of the cuticle collagen of C elegans, Bergerac strain."

Ouazana R

[Worm Breeder's Gazette, 1980]

The major components of the cuticle were found, in Ascaris des to be proteins (Bird 1956), the most important being collagen (Watson and Silvester 1959; Josse and Harrington 1964). In order to obtain this major protein, intact cuticles from C. elegans were isolated by sonication and incubation with 1% SDS, according to the method described by M. Kusch at the Woods-hole workshop. The purity of the preparation and the preservation of the in situ characteristics of the isolated cuticles were controlled by electron microscopy. Proteins represent about 65% of the dry weight of the isolated cuticles and collagen 70% of the proteins. Taking into account the fact that the cuticle is soluble in reducing solutions, as is the case for Ascaris (Evans et al. 1976), we prepared the C. elegans cuticle collagen by reduction with -mercaptoethanol in a buffer containing 8M Urea followed by carboxymethylation with sodium Iodoacetate. This collagen analyzed by SDS-Polyacrylamide gel electrophoresis gave seven bands (fig.). Their molecular weights are : 185000, 155000, 92000, 65000 (the predominant band) 51000, 32000, 20000 (the gamma, 11 and alpha1 components of Calf skin Type I Collagen were taken for calibration). All these components are sensitive to bacterial collagenase. The C. elegans cuticle collagen was submitted to molecular sieve chromatography and now to ion exchange chromatography on phosphocellulose to obtain purified components. [See Figure 1] The amino acid composition of the cuticular collagen of C. elegans and the fractions obtained by molecular sieve chromatography showed high hydroxyproline content (10%) and low proline content (13%) compared to Ascaris cuticle collagen (2% and 36% respectively, Evans et al. 1976). The proline and hydroxyproline contents are close to those found in vertebrate collagen. But like all known invertebrate cuticle collagen, C. elegans lacks hydroxylysine. The aim of the present work is to determine whether the separated components are genetically distinct chains and could represent different collagen types, or if these components all belong to the same type of collagen.

Bird DM et al. (1991) Worm Breeder's Gazette "A Model for the Structure of the dpy-13 Collagen"

Bird DM, Chen TY

[Worm Breeder's Gazette, 1991]

Using a combination of computer and hand alignments, we have generated a model for the dpy-13 product in which the peptide folds back on itself to form a triple helical structure with non-helical termini (telopeptides) and a region of potential interaction with other collagens. This model is consistent with a proposal based on a classical, physical biochemical analysis of Ascaris collagen (1) ( McBride and Harrington, Biochem., 6: 1484, 1967). Key features of our model are: 1) The four stretches of sequence able to form triple-helix define three domains (3, 5 and 7 in Fig. 1). Domains 5 and 7 are available to participate in helix formation along most of their length, but domain 3 is sufficient to interact with only the amino-half of 5 and the carboxy half of 7. Perhaps the hypervariable domain (2) interacts with the remainder of domains 5 and 7. Alternatively, another peptide (8) with a gly-X-Y motif might contribute to triple-helix. The discontinuity in 5 possibly is required for such interactions. This folding arrangement places the second gly-X-Y region (5) in anti-parallel to the other two (3 and 7), effectively reversing X and Y order. This reversal enhances the preferential distribution of the bulky residues in the X position (Traub, Israel J. Chem., 12: 435, 1974) and places six of the seven proline residues in suitable locations for X/Y pairing (Jones and Miller, JMB, 218: 209, 1991). 2) Rather than being formed from the termini of peptides, the telopeptides are generated by turns in the non-gly-X-Y regions (4, 6 in Fig. l) and stabilized by disulfide linkages; the location of cysteines is consistent with formation of disulfide bridges between the ends of 1 and 5, and start of 5 and the end of 7. Chou/Fasman algorithms predict alpha-helix at both turns. The distribution of lysine residues in these turns is characteristic of other telopeptides. . 3) Sequences towards the amino terminus are not shown as being involved in folding as they presumably include a signal sequence and pro-collagen domain that are absent in the mature protein. It is not inconceivable that the putative pro-collagen peptidase target is the 'Box A consensus' (Levy, et al., WBG 11(5): 36) in the center of the otherwise hypervariable region 2. 'Box A' also contains a putative glycosylation site. It has been suggested that the semi-dominant alleles of dpy-13 (e.g. e184) might have products with aberrant collagen (triple helix?) formation but normal sites of interaction with other cuticle components, whereas the products of recessive alleles (e.g. e225) might have normal collagen helix but an altered ability to interact with other components (von Mende and Riddle,WBG 11(4): 50). The molecular location of these defects is consistent with our folding model. 1. The size of collagen monomers calculated by these workers is not in accord with that predicted from C. elegans gene sequence data. We feel that the conditions used for their sedimentation studies (low concentrations of reducing agents) were conducive to cross-linking of monomers. [See Figure 1]

Fire A (1986) Worm Breeder's Gazette "a Drosophila heat shock promotor functions in C elegans."

Fire A

[Worm Breeder's Gazette, 1986]

In the last gazette, I reported that coinjection of one plasmid carrying the amber suppressor sup-7 (used as a selectable marker) with a second, unselected plasmid could be used to obtain stably transformed lines which contain sequences from the second plasmid in addition to sup-7 DNA. I have used this to introduce a plasmid pShZ2 ( from Sean Munro) carrying a Drosophila heat shock promoter (HSP) fused to sequences coding for E. Coli -galactosidase (see diagram). Of three independent stably transformed lines derived from these injections, two (CB4027 and CB4028) were analyzed on southern blots and found to contain the HSP- -gal fusion segment in apparently unrearranged form. -galactosidase activities from these transformed lines have been compared with control lines both by soluble enzyme assays and by histochemical staining. 1. SOLUBLE ENZYME ACTIVITIES: Worms were grown on a bacterial strain deleted for the -galactosidase gene , harvested as L4s and homogenized by sonication (painful on the ears) or by a new 'sandblasting' method (see below). The resulting lysates were incubated with ONP -Galactoside (yellow product) or 4- Methylumbelliferyl -Galactoside (fluorescent product) and enzyme activities determined spectrophotometrically. A very low basal level of enzyme activity was detected both in the parental line, and in sup- 7 transformed lines not carrying the HSP- -gal fusion. This basal level was not affected by heat shock (3 hours at 34 C). Under normal growth conditions (20 C) the two lines carrying the HSP- -gal fusion produce levels of -galactosidase similar to the parent line (within 2- fold). After heat shock, a striking increase of 10-20 fold in galactosidase level is seen with both CB4027 and CB4028. 2. HISTOCHEMICAL STAINING: Whole worms were fixed and stained for gal activity as described below. No staining was observed in the parental line (with or without heat shock) or in CB4027 or CB4028 growing at 20 C. After heat shock, strong and reproducible staining of both CB4027 and CB4028 was observed (>95% of the animals stain). The staining appears almost exclusively in the pharynx (all stages) and in embryos. The localization of the stain is unlikely to reflect a simple permeability difference, since squashed and/or cut animals show similar patterns of staining. Additionally, injection of pure enzyme into the gonad, gut or pharynx of wild type animals followed by fixation and staining as above resulted in specific staining in each case of only the injected tissue. Histochemical staining is being used to examine the segregation of the inducible galactosidase activity: Preliminary evidence suggests that multiple loci (2-3) in CB4027 carry functional HSP- -gal segments. In particular at least one locus unlinked to the amber suppression activity (which maps on chromosome IV(L) near unc-17) can confer heat inducible gal activity. This is consistent with estimates of copy number for the gene fusion, between 5 and 20 for the two strains. The possibility of extrachromosomal inheritance for the transgenic HSP- - gal loci cannot yet be ruled out but seems unlikely due to the DNA copy number and the fact that inducible gal activities have been maintained in the two strains in the absence of selection for over 30 generations. A rough estimate of the induced -Galactosidase as a fraction of total protein can be obtained either from soluble enzyme assays or from histochemistry (by comparison with injections of pure enzyme). These estimates on the order of .001% and .01% respectively (the difference may reflect losses in solubilizing the enzyme). Why is staining in induced animals limited to the pharynx? Obvious suspects include the nature of the worm heat shock response, the synthetic rates and stabilities of -gal protein and message in different tissues, and possible fortuitous expression signals in the HSP- -gal fusion plasmid that was used. In any case the strains may prove useful in finding nematodes on warm summer nights. SAND EXTRACT: To worms (10-100 l) in a 1.5ml microfuge tube are added about equal volumes of buffer and acid washed fine grain sand. The tube is then agitated vigourously-- I do this by striking the bottom of the tube along the pegs of a plastic (Gilson) test tube rack 10-20 times (like bad, loud guitar playing). Sand can be removed by centrifugation or filtration. All the worms are lysed and completely broken up by this procedure and recovery of total protein and enzyme activity appear comparable to sonication. -GAL STAIN: Wash worms in water and place in drop on a slide (I use 4-well multitest slides). Dry slides 5-10 min in a dessicator jar under vacuum (<2mbar). Dip the dried slides in acetone and dry on benchtop. Add 100 l of staining solution [0.2M NaPhosphate pH 7.5, 1. 0mMDTT; saturate with substrate (6Bromo2NapthylBDgalactoside from Sigma) and add 0.004% SDS] for 1 hr at room temperature. Then add 20 l of 0.125% 'fast blue B diazonium salt' [BDH], mix well, and staining should appear within 10-20 min. The substrate is cleaved by - galactosidase to yield a local insoluble precipitate of 6Bromo2Napthol; this reacts with the diazotized Fast Blue to form insoluble turquoise dye (Rutenberg et al J. Histochem. and Cytochem. 6, 122). The fast blue must be dissolved (H20) just prior to use or a nasty orange stain develops which can obscure the reaction product. Some staining near the mouth in induced live worms can be obtained using this protocol without drying and acetone steps. [See Figure 1]