Edwards MK (1987) Worm Breeder's Gazette "collagens, cuticles and chromosome V."

Edwards MK

[Worm Breeder's Gazette, 1987]

On the right side of the cluster on Chromosome V are several genes which, when mutated, cause defective cuticles. These genes include vab-8, rol-4, cuticles are comprised predominantly of collagens, I wanted to determine whether any of these genes do in fact encode a collagen. Using radioactivity-labeled pcol-2, I probed restriction digests of overlapping cosmids contained within the myo-3 contig which had been mapped to this region. (See maps below). Two of these cosmids, C12A12 and ZC348, cross-hybridized to pcol-2 under low stringency conditions. Interestingly, the HindIII fragment of ZC348 had the same molecular weight (3.5 Kbp) as did the HindIII fragment of pcol-1. As a further test for identity, I probed double digests of both ZC348 and pcol-1 (HindIII/BamHI and Hind III/HinFI) with labeled pcol-1 under high stringency conditions. Identical restriction fragments were seen for both clones. The localization of col-1 to this region is a tremendous aid to our study of cuticle-defective mutants: col-1 has been sequenced (Kramer et al., 1982, Cell 30, 599) and its pattern of developmental expression has been determined (Cox & Hirsh, 1985, Mol. Cell. Biol. 5, 363). We are now using the col-1 gene as a probe to look for RFLPs in mutant DNAs. To study the function of the col-1 gene product we plan to isolate specific antibodies which can be used for biochemical and morphological analyses of wild-type and mutant cuticles. [See figure 1]

Edwards MK (1981) Worm Breeder's Gazette "hybridization of DNA probes to RNA in situ."

Edwards MK

[Worm Breeder's Gazette, 1981]

In situ hybridization should prove useful for analyzing changes in RNA populations during C. elegans development without isolating biochemical quantities of similarly staged embryos or specific tissues. RNA can be detected in various fixed preparations with well- preserved morphology. Embryos, squashed according to the procedure of Gossettand Hecht (1979 C. elegans meeting), maintain their cell boundaries so that the number of cells, and thus the developmental age, is easily determined. Larvae and adults, fixed in ethanol/acetic acid (3:1), frozen in O.C.T. compound from Tissue-Tek, and sectioned at -20 C, retain good tissue morphology. With Nomarski optics, well- preserved muscle, gonad and intestinal tissues can be recognized, and with polarization optics, muscle birefringence and gut granules are visible. Alternatively, adults can be cut open in M9 such that the gonad and intestine extrude as separate tissues from the body of the worm. When squashed in 45% acetic acid, frozen,and then fixed in ethanol/acetic acid, these tissues retain good Nomarski morphology. After hybridization of a radioactive DNA probe to these preparations, followed by autoradiography, grain densities can be determined for particular embryonic cells or adult tissues. I can currently detect abundant RNAs using recombinant DNA probes. In situ hybridizations with cloned nick-translated DNAs containing ribosomal, histone, actin (from Jim Files), and myosin (from John Karn) genes produce strong signals (10 to 200 fold over background after 10- day exposure) under conditions in which non-homologous DNA (Charon 3) gives no signal over background. In addition, three cloned DNAs from our Charon 3 library that plaque-hybridize with C. elegans cDNA also hybridize to RNAs in situ. These three probes show differential hybridization between young embryos (1-50 cells) and old embryos ( comma to hatching) and between two adult tissues (gonad and intestine). Present work involves more precisely characterizing the amount of hybridization as functions of developmental time and tissue specificity for these and other clones.

Edwards MK (1982) Worm Breeder's Gazette "analysis of three messages by cytological hybridization."

Edwards MK

[Worm Breeder's Gazette, 1982]

Using the technique of cytological hybridization described here a year ago, I have analyzed RNA's complementary to three cloned DNA's from C. elegans genomic libraries: collagen, actin, and myosin. The collagen probe hybridizes specifically to the dorsal and ventral hypodermal cells in the adult. Collagen messages cannot be detected in the ovary or in early embryos. They are observed in later embryos beginning at 2 to 3 hours after the 2-cell stage at 25 C; their time of appearance corresponds to that of poly-A-containing message as determined by Hecht et al. (Devel. Biol. 83:374, 1981). Due to the conservation of actin coding sequences, the actin probe hybridizes to transcripts for muscle and cytoplasmic actins. Cytological hybridizations show that actin messages are abundant in the ovary and are stored in the egg as maternal RNAs. Additional expression occurs during embryogenesis and in adult body-wall muscle, pharynx, and spermatheca. In contrast with the actin probe, the myosin clone hybridizes primarily to transcripts of the myosin gene expressed in body-wall muscle. There is little cross-hybridization to other myosin transcripts. The most intense hybridization occurs in muscles, while little hybridization is seen in the intestine or ovary. In addition, the myosin probe does not hybridize to early embryos, but does hybridize strongly to older embryos undergoing morphogenesis and differentiation.

Edwards MK et al. (1987) International C. elegans Meeting "suppressors of lin-1."

Edwards MK, Clark SG, Horvitz HR

[International C. elegans Meeting, 1987]

Edwards MK et al. (1979) International C. elegans Meeting "in situ hybridization techniques."

Wood WB, Edwards MK

[International C. elegans Meeting, 1979]

Edwards MK et al. (1983) Worm Breeder's Gazette "suppressors of the multivulva mutation lin-1(e1275)."

Edwards MK, Horvitz HR

[Worm Breeder's Gazette, 1983]

The early steps in the determination and differentiation of the vulval precursor cells P(3-8).p have not been well defined genetically.

Edwards MK et al. (1990) Trop Med Parasitol "Antigenic and dynamic properties of the surface of Onchocerca microfilariae."

Busto P, James ER, Edwards MK, Carlow CK, Philipp M

[Trop Med Parasitol, 1990]

We analyzed the antigenicity and stability of the surface of skin microfilariae (mf) of Onchocerca cervicalis, a horse parasite. These mf express antigens on their surface that are cross-reactive with the cattle parasite O. lienalis and with the human parasite O. volvulus. The surface of living O. cervicalis mf was radioiodinated using Iodogen and the labeled components were solubilized in buffers containing sodium dodecyl sulphate (SDS), or extracted with the milder detergent octyl-beta-D-glucopyranoside (OGP). Electrophoresis of this material showed seven prominent bands, one of which (14 kDa) was specifically precipitated by antisera from rabbits immunized with mf from either O. cervicalis, O. lienalis, or O. volvulus, and by human sera obtained from infected individuals in Chiapas, Mexico. Other components were precipitated by either the rabbit or the human sera. In addition, antisera from mice immunized with O. cervicalis mf bound specifically to the surface of freeze-thawed uterine O. lienalis and O. volvulus mf as detected by immunofluorescence. This fluorescence was lost from the surface of O. cervicalis mf in a temperature-dependent fashion. Live mf incubated on ice with mouse anti-mf antisera and secondary FITC-GAM, showed uniform surface fluorescence. When these mf were incubated at 37 degrees C, but not at 0 degrees C, the fluorescent pattern changed with time. First, small non-fluorescent patches arose, followed by an increasingly wide belt devoid of fluorescence, and finally, no visible fluorescence. These changes in the mf surface suggest potential mechanisms for immune evasion by filarial parasites.

Edwards MK et al. (1991) International C. elegans Meeting "COLLAGEN GENES FLANKING MY0-3 (V)."

van der Keyl H, Edwards MK, Quisel A, Maloof JN

[International C. elegans Meeting, 1991]

We have identified two collagen genes linked to myo-3: col-? Iies approximately 80 kb to the left of myo-3, while col-l lies approximately 120 kb to its right. We have determined the DNA sequence of 1771 nucleotides which contains the coding region of the col- ? gene. A putative initiating met codon occurs at position 701 and is immediately preceded by a potential splice acceptor site (TTTTAGG) with no corresponding upstream donor site, suggesting that the co 1- ? message may have a trans-spliced leader. The coding region continues uninterrupted by introns to a stop codon at position 1646, predicting a 315 aa protein with an isoelectric point of 3.59 and a typical cuticle collagen structure. Comparisons of the three conserved cysteine-rich domains in col- ? with these domains in other C. elegans collagen genes reveal that this new collagen belongs in the col-7, col- 8, col-l 9 group. However, the COO- domain of col- ? contains 41 aa, while other members of this group have only 14 or 15 aa. A search for conserved DNA sequences in the S' flanking region of the gene reveal homologies with two repeats found upstream of most collagen genes and with one repeat found upstream of only dauer-specific collagen genes. Linked to myo-3 are four cuticle-defective loci (sqt-3, rol4, ali-l, lon-3). To determine which of these genetically defined loci are encoded by col-? and col-l, we have generated 500-600 bp PCR fragments of the COO- end of each gene from eight mutants. We have cloned these fragments and are currently sequencing them. A missense mutation (Gly to Glu) in the triple helical domain of col-l has been detected in DNA from sqt-3(sc63), although we need to verify that this G to A transition was not generated during the amplification or cloning.

Edwards MK et al. (1989) International C. elegans Meeting "structural and functional analyses of collagens linked to sqt-3 (V)."

Oke CV, Edwards MK, Maloof JN, Price L

[International C. elegans Meeting, 1989]

Edwards MK et al. (1983) Dev Biol "Location of specific messenger RNAs in Caenorhabditis elegans by cytological hybridization."

Wood WB, Edwards MK

[Dev Biol, 1983]

We have developed an autoradiographic technique for detecting specific Caenorhabditis elegans messenger RNA molecules in situ by hybridization of labeled, cloned DNA probes to fixed tissue sections and squashes of embryos and adults. We report analyses with probes of actin and collagen gene sequences from a C. elegans genomic clone library. Hybridization is RNase sensitive and tissue specific. In adults the actin probe, which recognizes cytoplasmic as well as muscle actin mRNA, hybridizes strongly to muscle and distal gonad (ovary), somewhat less strongly to maturing oocytes, and weakly to intestine. The collagen probe hybridizes weakly to distal gonad and intestine and very strongly to subcuticular tissues, in particular to the hypodermal cells and syncytial cytoplasm of the lateral hypodermal ridges, which are the sites of cuticle synthesis. In embryos, hybridization to squashes indicates that actin message is present at fertilization, decreases during early cleavage, and then increases again during morphogenesis. By contrast, collagen message is absent until the 100-cell stage and then increases rapidly during morphogenesis. The number of cells labeled is consistent with the view that the collagen probe hybridizes to hypodermal precursor cells. We estimate that our present methods can detect messages representing about 0.2% or more of the total mRNA population, and increases in this sensitivity should be possible. Therefore, the cytological hybridization technique should be useful for determining temporal and spatial patterns of specific mRNA distributions during development, at least for abundant and