Harfe BD et al. (1994) Worm Breeder's Gazette "ceh-24, a New NK Class Homeodomain Gene."

Okkema PG, Fire A, Harfe BD

[Worm Breeder's Gazette, 1994]

ceh-24, a new NK class homeodomain gene Brian Harfe 1, 2, Pete Okkema 1, and Andy Fire 1 1 Carnegie Institution of Washington, Department of Embryology, 2 Biology Graduate Program, The Johns Hopkins University The NK class of homeodomain proteins are a set of regulatory factors with a broad evolutionary distribution and a variety of expression patterns. Analysis of pharyngeal myosin regulation led to the identification of a member of this family, ceh-22, which appears to mediate aspects of pharyngeal gene expression (Okkema and Fire, Development 120, 2175). To determine the extent of the NK homeodomain family in C. elegans we have used a degenerate primer based PCR strategy. Among several candidates from the initial PCR, we have so far characterized one gene, denoted ceh-24. The CEH-24 homeodomain falls into the NK class, but is closer to diverse members of the NK family seen in other species than to CEH-22. In addition to the homeodomain, CEH-24 contains an acid activation domain C-terminal to the homeodomain. ceh-24 maps (thanks to Alan Coulson) to chromosome V just left of him-5.

Aamodt EJ et al. (1990) Worm Breeder's Gazette "Transformation of C."

McGhee JD, Aamodt EJ, Chung MA

[Worm Breeder's Gazette, 1990]

To identify the control regions of the C. elegans gut-specific esterase gene (ges-1), we are using a variety of approaches including a comparison of the upstream control regions of ges-1 and the homologous esterase gene from C. briggsae (cloned and sequenced from the Baillie library by Brian Kennedy and Fran Allen). For this approach to be valid, we needed to show that the esterase gene from one species is properly expressed in the other species. It was easy to show that the C. briggsae esterase is expressed in the gut of C. elegans by transforming the C. briggsae gene into a ges-1(0) strain of C. elegans and then staining the transformants for esterase. We did this experiment using both transient transformation and extrachromosomal heritable transformation. To show that the C. elegans properly expressed in C. briggsae, we needed to make a strain of C. briggsae that was transformed with the C. elegans tried the rol-6 dominant mutant gene and followed the transformation procedure of Mello et al. (WBG 11-1:18 and WBG 11-3:12). Roller strains of C. briggsae were easily obtained (the rachis of C. briggsae is even easier to hit than that of C. elegans). When the ges-1 gene was used in a cotransformation with the rol-6 gene all of the esterase expression was confined to the guts of the transformants i.e. the transformants looked exactly like wildtype C. briggsae. We then used isoelectric focusing gels on extracts of the transformed C. briggsae followed by staining of the gel for esterase activity to show that the C. elegans indeed being expressed. Thus, the C. elegans expressed in C. briggsae and it is expressed only in the gut. A deleted construct of ges-1 that contains only 521 base pairs 5' from the translation initiation codon is expressed in the pharynx muscles of C. elegans (E. Aamodt, M. Chung and J. McGhee, manuscript submitted). When this deleted ges-1 gene was transformed into C. briggsae, the transformants also express the esterase gene in their pharynx. In other words, not only is the intact gene properly expressed but the ectopic expression of the deleted ges-1 is identical between the two species. Thus, both ges-1 and the mutant rol-6 genes are properly expressed in C. briggsae. Cotransformation of C. briggsae using the rol-6 marker gene should be useful to others who wish to see if their favorite C. elegans gene works in C. briggsae.

Crew JR et al. (1998) Worm Breeder's Gazette "The bli-1 and bli-2 genes encode collagens involved in strut assembly."

Crew JR, Kramer JM

[Worm Breeder's Gazette, 1998]

The adult cuticle of Caenorhabditis elegans consists of cortical and basal layers connected through a medial layer by struts. The medial layer and struts are unique to the adult cuticle. The struts are periodically placed within the cuticle, lying roughly in rows along either side of the annulae, oriented perpendicular to the plane in which the rest of the cuticle is arranged. The bli-1 and bli-2 genes have been physically and genetically mapped. Transformation rescue has been used to show that the bli-1 gene corresponds to sequence CO9G5.6, which encodes an unusual cuticle collagen. The amino- and carboxyl-terminal domains of this protein are roughly 400 amino acids, approximately 10 times larger than the corresponding domains in most cuticle collagens. These terminal domains contain large proportions of proline and charged residues. Although no known motifs or domains implicated in protein interactions are evident in these terminal domains, the large size and number of charges found in these domains imply that BLI-1 may connect the layers flanking the medial layer by interacting with other cuticle components. The mutant phenotype of bli-2 is rescued by cosmid C32E11, which contains an open reading frame encoding a more standard cuticle collagen. The mutant phenotype of these genes is cuticles covered by fluid-filled swellings. We have made double mutant combinations of bli-1 and bli-2 with several heterochronic genes and observed that such combinations yield blisters only when an adult cuticle is present. Using a temperature-sensitive allele of bli-2, we showed that the function of its product is necessary in the late L4 stage, the time during which the adult cuticle is synthesized. In addition, mRNAs for the bli-1 and bli-2 genes are expressed highly only during the L4 stage of development (B. D. Ackley and J. M. K., unpub.). These data together imply a defect specific to the adult cuticle. Transmission electron microscopy of bli-1 and bli-2 mutants shows that the struts are absent from the medial layer, which is filled by granular electron dense material or morphologically abnormal struts, implying that these proteins are involved in strut construction. The hypodermis of these mutants can vary in width and can also contain large membrane bound structures. These may be due to secretory defects caused by incorrectly processed collagens, as have been noted previously in vertebrates and for collagen type IV in the worm. Some bli-1 and bli-2 mutant animals have abnormalities in the three-pronged adult alae, such as four-pronged alae or gaps and small discontinuities in the treads. Because tread assembly is controlled by the lateral seam, we have examined its integrity. Animals mutant in bli-1 and bli-2 have normal numbers of seam cells that appear to fuse correctly. Some mutant animals also exhibit irregularities in the spacing of the annulae. It is thought that the circumferential actin bundles that arise in the hypodermis prior to each molt may aid in spacing these indentations. We have examined these actin bundles in animals undergoing the L4-adult molt; there do not appear to be any abnormalitites in bundle orientation or distribution in mutant animals. Blistered animals can have greatly shortened lifespans (3-4 days) and reproductive capabilities due to the severity of the blistering. We have observed that blisters can "heal" in a small percentage of animals, allowing them to live essentially a normal lifespan. Although the severity of the blistering phenotype differs between alleles, the frequency of healing events observed is about 10% in every case. We are unsure how this healing occurs, but it may involve the fusion of the cortical and basal layers. Mutations in bli-1 and bli-2 exhibit allele-specific, partially penetrant intergenic non-complementation, suggesting that their gene products may interact. We have also found similar interactions with the other Bli genes. In addition, we have TEM evidence that the medial layer is the layer affected in animals mutant in all of the Bli genes. These data lead us to believe that these genes may constitute the proteins necessary for constructing struts. Therefore, this system could be a valuable one for understanding the assembly and maintenance of a discrete extracellular structure.

Bektesh SL et al. (1987) Worm Breeder's Gazette "trans-splicing in C elegans."

Bektesh SL, Hirsh DI, Van Doren K

[Worm Breeder's Gazette, 1987]

Recently, an alternative form of RNA processing (transsplicing) has been shown to occur in C. elegans (Krause and Hirsh, Cell 49, 753-761, 1987). In this RNA processing reaction, sequences from one RNA molecule are joined in an intermolecular reaction to sequences in a second molecule. mRNAs encoded by three of the four actin genes in C. elegans were shown to contain the same 22 nucleotides at their 5' ends as a result of transsplicing. This sequence has been named the splice leader. If the mechanism is trans-splicing, then by analogy with trypanosomes, the two precursor RNA molecules would be joined through a Y-branch intermediate in an intermolecular reaction. Our experimental results indicate that a Y-branch intermediate is involved that can be cleaved with purified debranching enzyme isolated from vertebrate cells; therefore, trans-splicing is indeed the mechanism in C. elegans.We were interested in characterizing other mRNAs that acquire the splice leader. A cDNA library was constructed from poly A+ mRNA and screened using an oligonucleotide complementary to the splice leader. More than 100 clones were isolated and about two dozen have been characterized and sequenced. In an attempt to identify these clones as having homology to known genes, the DNA and deduced amino acid sequences were searched against existing databases. Two clones have strong homology with two different ribosomal protein genes from rat and yeast and are therefore thought to encode ribosomal proteins in the nematode. A second approach has been to examine published nucleotide sequences of C. elegans genes for the presence of a splice acceptor sequence. If such a sequence is present 5' of the initiator ATG codon, and no splice donor sequence is found farther upstream, an oligonucleotide homologous to a portion of the coding sequence is constructed and used for primer extension sequencing of the mRNA. In this way, the mRNAs coding for ubiquitin and glyceraldehyde-3-phosphate dehydrogenase have been shown to contain the splice leader at their 5' ends. Not all mRNAs in C. elegans acquire the splice leader. Two approaches were taken to determine what proportion of the messages in C. elegans acquire the splice leader. First, quantitative dot blots were done using poly A+ mRNA and oligonucleotide probes. Following hybridization, samples were quantitated in a scintillation counter and compared to poly A+ mRNA hybridized to an oligo dT oligonucleotide as a measure of all poly A+ mRNAs. This work suggests that approximately 10% of the poly A+ mRNAs in the worm acquire the splice leader. A second approach asked what proportion of the translated proteins in an in vitro reaction are encoded by mRNAs containing the splice leader at their 5' ends. Poly A+ mRNA was translated in a rabbit reticulocyte in vitro translation system either with or without the addition of an oligonucleotide complementary to the splice leader. The products of the reactions were separated by two-dimensional gel electrophoresis. Approximately 10% of the proteins translated in vitro were arrested upon the addition of the oligonucleotide complementary to splice leader illustrating that these proteins are the products of mRNAs containing the splice leader. Acquisition of splice leader does not appear to be developmentally regulated. Northern analysis of RNA isolated from oocytes (generously provided by Brian Kennedy and Jim McGhee) and from several larval stages suggests that the splice leader is present on mRNAs in general, and on actin mRNAs in particular, throughout development. Is this splice leader present in other organisms? Northern analysis of RNAs from other nematodes (C. elegans var. Bergerac, C. briggsae, Panagrellus revividus, Haemonchus and Ascaris) revealed that these worms contain the same or a very closely related splice leader. However, Northern analysis of RNAs from yeast, trypanosomes, Drosophila, Dictyostelium, Xenopus and human revealed that the evolutionary conservation of this splice leader sequence does not extend to these organisms.