Graham RW et al. (1990) Worm Breeder's Gazette "Rapid Amplification and Analysis of Ubiquitin cDNA Sequences from C. elegans, C. briggsae etc. etc. using Versatile Degenerate Ubiquitin Primers"

Candido EPM, Graham RW

[Worm Breeder's Gazette, 1990]

Based on Northern analysis with 3'-end specific probes we have determined that C. elegans must possess at least two classes of ubiquitin genes. We have cloned the major ubiquitin gene, UbiA, which encodes worm polyubiquitin, and have undertaken various schemes for the isolation of the non-UbiA member(s) of the ubiquitin family. Unfortunately, exhaustive screening of available C. elegans cDNA and genomic libraries and attempts to directly clone size-fractionated genomic DNA have led only to the reisolation of UbiA clones ad nauseum.

Klass MR et al. (1982) Worm Breeder's Gazette "repression of the spermatogenic pathway in the hermaphrodite."

Herndon M, Klass MR

[Worm Breeder's Gazette, 1982]

Using a combination of 2D gels and Northern blot analysis we have been able to demonstrate that the regulation of the spermatogenic pathway in the hermaphrodite occurs at the level of transcription at least with respect to the major sperm protein 15K. 2D gel analysis of pulse-labeled synchronous cultures of N2 shows that 15K is not synthesized in L1 or L2 larvae but is synthesized in L4 larvae during spermatogenesis and is then repressed after sperm production has stopped in gravid hermaphrodites. Northern blot analysis using our cloned genomic probe for 15K mRNA has demonstrated the lack of 15K mRNA in L2 and gravid hermaphrodites and its presence only during spermatogenesis in the hermaphrodite.

Plenefisch JD et al. (1987) Worm Breeder's Gazette "the lethal phenotype and temperature sensitivity of dpy-27 and dpy-28."

Meyer BJ, Plenefisch JD

[Worm Breeder's Gazette, 1987]

Mutations in dpy-27 and dpy-28 affect the viability of XX but not XO animals. In addition, these mutations disrupt dosage compensation resulting in XX but not XO animals over-expressing their X-linked genes (as assayed by Northern analysis [Meyer and Casson, Cell 47:871 1986] and by morphogenetic assay [DeLong, et al. Genetics, in press]).

Nilsen TW et al. (1988) Worm Breeder's Gazette "trans-splicing in Ascaris."

Bennett KL, Nilsen TW, Faser C

[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.

Snutch TP et al. (1984) Worm Breeder's Gazette "detection of coding regions in C elegans."

Baillie DL, Snutch TP

[Worm Breeder's Gazette, 1984]

A number of C. elegans workers have demonstrated the feasibility of utilizing RFLDs as molecular genetic probes for cloning and mapping genomic DNA sequences (Emmons et al, 1979; Rose et al, 1982). In addition, Sulston et al (Newsletter vol. 8, no. 2) have begun the herculean task of mapping the entire C. elegans genome. A question that arises from these efforts is how to identify the cloned sequences which are of interest - ie. coding sequences. The standard techniques of S-1 mapping, R-looping, Northern blotting and screening of cDNA libraries are labor intensive and too time consuming for the initial screening of large regions of cloned DNA. We believe that this problem can be simplified by utilizing the hybridization of cloned C. elegans probes to genomic blots of C. briggsae DNA. Evidence reported by Emmons et al (1979) and our own both indicate that while some portions of the C. briggsae and C. elegans genomes hybridize to each other, the majority of sequences have diverged significantly and do not cross-hybridize under moderate stringency conditions. Since C. elegans and C. briggsae are nearly identical, and in fact, are considered twin species (Dougherty, 1953), it is reasonable to assume that the divergent regions include mostly noncoding DNA sequences while the highly conserved regions code for the proteins necessary for nematode growth, development and reproduction. Our own results seem to support this idea. In the course of our work on C. elegans heat shock genes we have found that essentially only the cloned regions which we have found to react positively with RNA also show conservation to sequences within the C. briggsae genome. The condition that we use are probes nicktranslated to a specific activity of 1-2x10+E8 cmp/ g hybridized at 68 C in 5 x SSPE, 0.2% SDS and 2.5 x Denharts for 24 hours and then washed 4 times 20 minutes in 0.2 x SSPE, 0.2% SDS at 68 C. We have tested about 60 kb of cloned DNA around three different C. elegans regions which show homology to the Drosophila hsp70 gene. Two regions about 3 kb in size each are conserved between C. elegans and C. briggsae under these stringency conditions. From both Northern blots and the screening of B. Myers cDNA library we have found that these regions are coding. The third region of hsp70 homology does not react positively with RNA blots nor can we find any corresponding cDNAs in the cDNA library. In addition, none of the flanking regions of any of these regions reacts with RNA, shows positives from the cDNA library or cross-reacts with the C. briggsae genome. We believe that the initial blotting of cloned DNA to C. briggsae will give a good indication of whether the DNA is coding or not. We normally expose the filters for 24-36 hours with a screen. On longer exposures minor homologies between the two species appear but these are easily distinguishable from the coding regions which give good, strong signals.

Benian GM et al. (1984) Worm Breeder's Gazette "a proposal for cloning genes by Tc1 tagging of mRNA."

Moerman DG, Benian GM, Waterston RH

[Worm Breeder's Gazette, 1984]

In the course of our studies on unc-22 mRNA (see elsewhere in this issue) we made an unexpected observation--Tc1 can be incorporated into a full length relatively stable transcript. This in turn has suggested a potentially powerful method for recovering DNA from genes suffering Tc1 insertions. In studies of the mRNA from the strain RW7012, which contains unc-22( st136::Tc1) in a largely Bristol background, we found by Northern analysis that unc-22 hybridizing sequences were present at near normal levels but at a slightly lower mobility than the comparable wild type band. A similar Northern probed with Tc1 reveals the same band in RW7012 but does not hybridize to any comparable band in N2. Apparently the Tc1 is incorporated at least in part into the unc-22 message and accounts for the change in mobility. The relative stability of the composite message is curious and at this point unexplained. If this feature proves to be a frequent property of Tc1 induced alleles, it may be possible to use this feature to provide a short cut to cloning a gene. In the Northern of RW7012 RNA, only the unc-22 transcript gave a strong signal with the Tc1 probe. Using Tc1 as a primer against total or size fractionated RNA one could generate cDNA to the 5' side of the Tc1, which could in turn be used to identify cloned sequences in an appropriate library. We are currently trying to determine the generality of our findings by examining RNAs from various Tc1 induced alleles of unc-22 and other genes. The report by Levis, et al., (Cell 38:471, 1984) that P can be incorporated into a larger reasonably stable white composite message in Drosophila gives us hope that a reasonable number of Tc1 induced alleles will display read-through transcription of Tc1.

Huang X-Y et al. (1988) Worm Breeder's Gazette "a new trans-spliced leader in C elegans."

Huang X-Y, Hirsh DI

[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.

Blumenthal TE et al. (1994) Worm Breeder's Gazette "C. elegans U2AF 65."

Zorio DAR, Lea K, Blumenthal TE

[Worm Breeder's Gazette, 1994]

C. elegans U2AF65

Starr TVB et al. (1990) Worm Breeder's Gazette "A CANDIDATE SEQUENCE FOR THE GENE S-ADENOSYL-L-HOMOCYSTEINE HYDROLASE IS LOCATED NEAR THE DPY-14 GENE"

Rose AM, Starr TVB, Baillie DL

[Worm Breeder's Gazette, 1990]

Using the technique of cross-species hybridization to C. briggsae we have initiated a search for coding elements contained within seven cosmids located near dpy-14. Fragments detecting conserved regions between C. elegans and C. briggsae were used to probe Northern blots and screen cDNA libraries. A total of ten conserved fragments detected seven cDNAs. To better align the genetic and physical maps of this region the conserved fragments, cDNA clones, the breakpoint of the deficiency hDf8, and the probes identifying sP1 and hP9 have been positioned on a restriction map of the region. Two cDNA clones, sP1-1 and sP1-3, also detected conserved bands between C. elegans and mammals. Partial cDNA clones of both were sequenced and the sequence data used to screen the EMBL sequence data base. The sP1-1 sequence did not detect any significant matches however, the sP1-3 sequence detected sequence similarities with the gene for S-adenosyl-L-homocysteine hydrolase (AHH) from rat(1) and D. discoideum(2). A comparison of the deduced amino acid sequence indicated that 60% of the amino acids between sP1-3 and AHH are identical; and, if conservative changes are considered the homology between sP1-3 and AHH is greater than 80%. Thus the high degree of similarity between sP1-3 and AHH suggests that the sP1-3 locus encodes the gene S-adenosyl-L-homocysteine hydrolase.

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.