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[
Worm Breeder's Gazette,
1992]
Background. The extracellular matrix (ECM) plays an important role in maintaining the structural integrity and cellular functions of a multicellular organism. ECM components, including collagens, fibronectin, laminin, vitronectin, tenascin, entactin, and proteoglycans, have been identified and characterized in mammalian tissues. However, the structure and organization of these components in the intact matrix remain unclear. The inability to isolate mutants that are defective in a single ECM component in higher organisms impedes this type of analysis in vivo. With the advantage of the well-characterized genetics in C. elegans, we initiated a project to study the organization and structure of ECM using C. elegans as a model system. Our approach was to generate and characterize a panel of monoclonal antibodies against C. elegans ECM to use as markers for structural studies including immunofluorescent microscopy. In the future, we plan to use these monoclonal antibodies in affinity chromatography for the purification of ECM components and to use as probes for isolating cDNA. Experimental Approach. The ECM components were extracted from a mixed population of adult and juvenile C. elegans using the procedure shown in Figure 1. [See Figure 1] BALB/c mice were immunized intraperitoneally with 100 g of ECM extract homogenized in complete Freund's adjuvant followed by three bi-weekly injections of 50 g of ECM extract in incomplete Freund's adjuvant. Spleen cells from immunized mice were fused with myeloma cells and plated at a concentration of 2.5x10 +E5cells/well. Results. Of the 1,200 wells screened.approximately 32% of the hybridoma supernatants tested positive in ELISA against C. elegans ECM extracts. Supernatants from positive wells were tested by immunoblotting against C. elegans ECM extracts and by immunofluorescent microscopy on whole or fragmented C. elegans Protein species ranging from 20,000-180,000 daltons were detected by the hybridoma supernatants in immunoblotting, and supernatant recognition ranged from complex patterns of multiple bands to a single band. Immunofluorescent studies also revealed diverse staining patterns, which included staining between muscle and hypodermis, around muscle bundles, in layers surrounding intestines and gonads, and around the cuticle. Seven of the hybridomas were purified by limiting dilution cloning and injected into mice for ascites tumor production. We would like to make these monoclonal antibodies available to interested investigators in the near future. (This project was part of a course, Biotechnology Laboratory: Molecular Recognition (BSC 352), which was composed of 70% graduate students and 30% undergraduate students, and was supported by the Department of Biological Sciences and the College of Arts and Sciences at Illinois State University. We also would like to thank Robert Barstead for providing worm fragments and helpful discussion.)
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[
Worm Breeder's Gazette,
1994]
Somatic Regulation of Germ-line Development, Part II; Non-Autonomy of Hermaphrodite Germ-line Sex Determination Jim McCarter and Tim Schedl. Dept. of Genetics, Washington Univ. School of Medicine, Sl. Louis, MO 63110, jim@wugenmail.wustl.edu
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[
Worm Breeder's Gazette,
1988]
While determining the 5' ends of C. elegans GAPDH mRNAs by primer extension sequencing, we found a new trans-spliced leader (SL2). The previously identified SL is present on mRNAs from three of the four C. elegans GAPDH genes. However, a new 22-nucleotide spliced leader sequence is found on mRNA from one of the four GAPDH genes. RNA Northern analysis showed that the SL2 is present on many RNAs isolated from C. elegans var. Bristol, C. elegans var. Bergerac and C. briggsae, but not on RNAs from Panagrellus redivivus and Haemonchus contortus. In this regard it differs from the original SL that is found in other nematodes. The hybridized RNAs showed a broad spectrum of different molecular weights. From the genomic Southern blot, it seems there are two clusters of SL2 genes. We have screened a C. elegans EMBL4 genomic library. Two groups of phages were isolated. Using the phage DNAs as templates, two kinds of SL2 genes have been sequenced. These two genes are nearly identical in the SL RNA region but differ in the 5' and 3' flanking regions. A consensus Sm binding site of snRNAs is present. The SL2 RNA can be folded into a secondary structure similar to that proposed for the original SL RNA. The SL2 RNA probably has a TMG cap because it can be precipitated by anti-TMG-antibody. The original SL and the new SL2 are similar; 16 out of 22 nucleotides are the same in the best alignment.
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[
Worm Breeder's Gazette,
1989]
We have previously reported the isolation and characterization of a cDNA clone corresponding to the 4.6 Kb transcript of
tra-2 (1). Primer extension and S1 protection analyses of the 5' end strongly suggested that this
tra-2 transcript was trans-spliced. Sequence ambiguities impeded identification of the leader by dideoxy sequencing RNA using reverse transcriptase. Consequently, we have used a PCR method (2) to obtain additional cDNA clones that encompass the entire 5' end of the 4.6Kb transcript. In brief, the 4.6Kb transcript of tra- 2 is trans-spliced to the SL-2 sequence originally found associated with the C. elegans gene, GAPDH (3). Surprisingly, we have also isolated 5 other cDNA clones that contain variations in the SL-2 sequence. These differences include insertions and deletions that result in a 23 or 21 nucleotide leader, and base substitutions in the original SL-2 sequence. We do not know if these differences are artifacts induced during the PCR reaction or if they reflect heterogeneity among SL-2 genes. If the latter, then this observed heterogeneity could explain our inability to identify the
tra-2 leader by reverse transcriptase sequencing. These results also imply that a transcript can be trans-spliced to leader sequences encoded by different genes, perhaps with different regulatory functions.
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[
Worm Breeder's Gazette,
1988]
In the last issue of the Gazette (Vol. 10, no. 1), we reported that RNAs from many species of nematodes have the same (or closely related) spliced leader as C. elegans. In C. elegans the gene encoding the SL precursor (preSL) is found on a 1 kb tandem repeat of ~110 copies that also contains the 5S rDNA (Krause and Hirsh, Cell 49:753-761, 1987). However, approximately 6 of the preSL genes are located on DNA fragments outside the 1 kb repeat. These copies are present both with and without the 5S rDNA genes. We examined the genomic organization of the preSL genes in Panegrellus redivivus and Haemonchus contortus by Southern blot analysis and screening genomic libraries. Both nematodes have many copies of the preSL gene. In P. redivivus there does not appear to be a preSL gene repeat; however, we have identified two 5S rDNA repeats, but preSL genes are not associated with these repeats. Some of the preSL genes are associated with 5S rDNA genes which are outside the repeats, while other preSL genes are independent of 5S rDNA. In H. contortus it is possible that a portion of the preSL genes are on a repeated fragment or are clustered. In contrast to C. elegans and P. redivivus, the preSL genes in H. contortus are not associated with 5S rDNA genes in repeat units or outside a repeat unit. We have isolated and sequenced eight preSL genes from genomic libraries of P. redivivus and H. contortus and compared the sequence to the C. elegans preSL gene. There are several regions of sequence conservation among the preSL genes from these three genera of worms. The 22 nt SL sequence is exactly conserved, as is the GT splice donor following the SL in seven of eight preSL genes, the three nucleotides immediately preceding the SL are conserved. It is possible that these three nucleotides are actually part of the SL but have gone undetected due to post-transcriptional modifications. In addition, an Sm binding site is found in position 65-70 in seven of eight of these genes. The gene that lacks the three conserved nucleotides preceding the SL is the same gene that lacks the Sm binding site. Therefore, we suspect that this may be a pseudogene. We are in the process of determining which of the P. redivivus and H. contortus preSL genes are expressed and what sequences outside of the preSL genes have been conserved.
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[
Worm Breeder's Gazette,
1990]
The trans-spliced RNA leader might play a role in regulating the translation and/or stability of SL-containing mRNAs. This possibility led us to seek transacting factors that might mediate this regulatory function. Protein factors from C. elegans cells (either embryos or worms from mixed stages) that bind with high specificity to the trans- spliced RNA leader sequence (SL) have been identified. Whole cell extracts were shown by a mobility shift electrophoresis assay to form two RNA-protein complexes involving the SL1 sequence. Complex formation could be abolished by preincubation with excess, unlabeled competitor SL1-containing RNA (which is the 5' non-coding region of actin-1 mRNA fused to the firefly luciferase coding region) but not by nonspecific, control RNA that did not contain an SL1 sequence. Spliced leader binding protein-1 (SLBP1) is distinct from SLBP2. The binding of these proteins to SL1 is cap dependent though independent of the detailed structure of the cap; either monomethyl- or trimethyl guanosine cap stimulates binding. We are purifying SLBP1 and SLBP2. Future work will be focused on the characterization of these proteins and elucidation of their biological functions.
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[
Worm Breeder's Gazette,
1990]
A subset of C. elegans mRNAs receives a 22 nucleotide leader from a precursor RNA called SL RNA. This RNA has the hallmarks of a typical snRNA: it is bound to the 'Sm' proteins and it has the unusual cap nucleoside, trimethylguanosine (TMG). Because this cap is on the 5' end of SL RNA it is transferred to recipient RNAs, along with the 22 nucleotides, during trans-splicing. Originally we believed this unusual cap was subsequently altered or removed, but it isn't. Since translation in eukaryotes is initiated by binding of an initiation factor, eIF-4E, to the cap (normally monomethylguanosine) the presence of a different cap on some mRNAs could have important functional consequences. For this reason (and also because a reviewer suggested it might be a good idea) we undertook to find out whether these TMG-capped mRNAs are found on polysomes. We made polysomes from young adult worms by a procedure adapted from a method for making plant polysomes. The procedure is briefly outlined below. We then used anti-TMG antibodies to demonstrate that actin-1 and actin-3 mRNAs found in the polysome preparation are indeed TMG capped. We conclude that TMG-capped mRNAs are translated in C. elegans. Several mechanisms by which translation may be initiated on these mRNAs are possible: 1. C. elegans eIF-4E might recognize both kinds of cap structure.
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[
Worm Breeder's Gazette,
1991]
We have isolated from a soil sample, in Sao Paulo - Brazil, a rhabditid nematode which we are characterizing at the morphological, biological and molecular levels. The isolate we are studying is temporarily being called B6/D6 until species and genus designations can be confirmed. C. elegans and B6/D6 probably belong to two different subfamilies of the Rhabditidae. Our object in these studies is to characterize a species sufficiently far from C. elegans such that only highly selected sequences have been maintained, but sufficiently close that unequivocal sequence alignments can be made. We chose B6/D6 because it is morphologically very similar to C. elegans, but so distant that the C. elegans vit genes fail to cross- hybridize with B6/D6 DNA at low stringency. The first genes we have studied are those coding for the SL RNA, the donor in trans-splicing. By Southern blot we have shown that the SL1 RNA genes of B6/D6 are dispersed throughout the genome and that they are not physically linked to the 5S rRNA genes. This is in contrast to what has been shown in C. elegans where the SL1 RNA genes are physically linked to the 5S rRNA in a single cluster in the genome (1). The organization of B6/D6 SL1 RNA genes is more like what has been found in Onchocerca volvulus, a parasitic nematode of man (2). We have isolated and sequenced two B6/D6 SL1 RNA genes from a genomic library. At the primary sequence level there are differences at 17 out of 99 positions within the transcribed portion of the genes, whereas the 5' flanking regions appear unrelated to one another. The predicted SL RNA products are only distantly related to the C. elegans SL1 RNA, but they can adopt the characteristic SL RNA secondary structure, and the SL sequence itself is identical to the SL1 sequence found in all nematodes examined to date. Besides the primary sequence divergence between C. elegans and B6/D6 SL1 RNAs, there are two other findings which suggest these two species are quite distant from one another. First, neither B6/D6 SL1 RNA gene contains a sequence in its upstream region related to the highly conserved proximal sequence element of the C. elegans snRNA genes (3). Second, we have failed to detect any signal when an SL2 oligonucleotide was used to probe both the B6/D6 genomic Southern blot and library. This result suggests that SL2 may be restricted to the Caenorhabditis genus or at least to the Peloderinae subfamily. We hope that B6/D6 may be of use to researchers performing sequence comparisons to help identify important regions. We have also cloned the B6/D6
vit-6 homolog. So far we have sequenced a third of this gene. The B6/D6 protein is 43% identical to the C. elegans vitellogenin, and intriguingly, there are some different asymmetries in codon usage. Supported by FAPESP, CNPQ & USP-BID program
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[
Worm Breeder's Gazette,
1988]
It has been reported that Ascaris RNAs contain a splice leader sequence similar to that of Caenorhabditis tesh et al., WBG 10-1:67). When we first sent Ascaris des RNA samples to the laboratory of D. Hirsh, we also tested them with an oligonucleotide corresponding to the C. elegans spliced leader (SL) and found, as his group did, that a similar splice leader is present in Ascaris. Recently one of us (T. N.) has shown that the same 22 nt leader is present in the parasitic nematode causing human lymphatic filariasis, Brugia malayi (P.N.A.S., in press). Because Ascaris offers potential advantages to do biochemistry not available in Brugia, we have recently collaborated in further characterizing trans-splicing in Ascaris and in cloning the Ascaris leader sequence. As in C. elegans, we have found by Northern blot analysis that a small (~100nt) polyA-RNA species is recognized by the spliced leader, and multiple mRNAs are detected when polyA+ RNAs are used. All RNAs tested by Northern analysis including early embryonic (<30 cell), larval, oocyte and gut were positive, implying, but certainly not proving, no tissue or stage-specificity. By Southern blot at least two enzymes, ScaI and HaeIII, cut genomic DNA to an ~1 kb repeat. The same pattern of digestion was seen using the 5S ribosomal gene probe, indicating the trans-spliced leader is located in the 5S repeat, as found in C. elegans and in Brugia. An Ascaris genomic library constructed in lambda EMBL4 (Bennett and Ward, Dev. Bio. (1986) 118:141) was screened with the 22 mer SL oligonucleotide and with an oligonucleotide from the Brugia 5S genes. Positive lambda clones which contained the 5S gene and the sequence leader have been plaque purified; one has been subcloned and sequenced in part. We have found the Ascaris 22nt sequence leader is exactly the same as the C. elegans and Brugia one, with the rest of the small RNA sequence divergent. To determine the percentage of messages that contain the spliced leader, we have used polyA+ RNA from a mixed C. elegans population and from various Ascaris tissues and developmental stages. With 0.5 g of C. elegans polyA+ RNA, the SL oligonucleotide hybridizes to 10.6% as much RNA as does a labeled oligo dT probe. In the same assay using Ascaris RNAs, only 1.1-2.0% of the messages appear to contain the spliced leader in gut, oocyte and larval RNAs. While this one experiment implies that fewer messages are trans- spliced in Ascaris, the results could also be due to Ascaris messages having much longer polyA+ tails than C. elegans. However, our Northern blots also imply fewer Ascaris messages are trans-spliced when Ascaris and C. elegans polyA+ RNAs are compared.
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[
Worm Breeder's Gazette,
1986]
Comparison of DNA sequences surrounding six C. tone genes reveals a novel conserved sequence located approximately 80 nucleotides upstream of the translational start site. The six genes include all of the genes in one genomic cluster (one of each of the four core histone genes), and two from another, unlinked cluster (one H2A gene and one H4 gene). All of the conserved sequences 5' of each of these genes are presented in the figure below. Starting at the ATG codon and moving upstream, they include a sequence adjacent to the translational initiation site typically found in eukaryotic mRNAs, a conserved sequence surrounding the transcriptional initiation site similar to one found in some sea urchin histone genes, a TATA box, and the novel sequence. With the exception of yeast, upstream consensus sequences in histone genes of other organisms are restricted to the genes for individual classes of histone proteins (i.e., they are H4 gene-specific or H2A gene-specific, etc.). In yeast a conserved sequence of 16 nucleotides activates transcription of histone genes in a cell cycle-dependent manner (Osley, M. H., Gould, J., Kim, 5., Kane, M. and Hereford, L. (1986) Cell 46, 537-544). Like the C. , this 16-mer is found upstream of different classes of histone-encoding genes. In the figure, numbering of nucleotides begins at the first translated nucleotide, and the number of non-matching nucleotides between each conserved region is given. The asterisk indicates the site of transcriptional initiation in two of the genes (+/- 1 nucleotide) determined from Sl mapping experiments. The arrow over the consensus transcriptional initiation sequence indicates the direction of transcription.