[
Worm Breeder's Gazette,
1980]
We are studying the C. elegans genes coding for actin and procollagen using as probes cDNA clones of Dictyosteleum actin (Firtel) and chicken procollagen (Doty). We have hybridized P-32 labeled Dictyosteleum actin DNA to Southern blots of various restriction digests of whole genome C. elegans DNA. The Dictyosteleum actin DNA hybridized strongly to a small number of bands, four in most restriction digests. From a collection of recombinant charon 10 phage carrying C. elegans DNA, we have isolated several clones complementary to the Dictyosteleum actin DNA. Two of these clones have been characterized. One clone, Act-1, contains a 17 kb insert of worm DNA and has two actin coding regions. As seen by hybridization of Dictyosteleum actin DNA to restriction fragments of the clone. This clone contains a 1000 base inverted repeat sequence, with the two copies of the repeat separated by about 2500 bases. This is visualized in the electron microscope as a snapback of denatured clone Act-1 DNA. Since the repeated sequences are in the same positions as the actin coding regions, they most likely represent two actin genes in a head-to-head ( or tail-to-tail) orientation. R-loop experiments are in progress to verify this conclusion by determining the exact positions of the genes, as well as to check for possible intervening sequences. A second clone, Act-2, with an insert of 14 kb, contains a single actin gene. Thus, three separate actin genes have been identified so far. One additional actin sequence, seen on Southern blots, is yet to be identified on a clone. We have purified actin mRNA from C. ps with clone Act-1 DNA and by DNA-cellulose affinity chromatography using cloned C. A. We have hybridized P-32 labeled cloned chicken procollagen DNA to Southern blots of various digests of C. ding a large number of bands hybridizing to the probe, some strongly and others with decreasing intensity. (Fifteen bands are seen with Eco RI digested C. e cut the cloned procollagen DNA with restriction enzymes into 3 pieces, isolated them, and hybridized each separately to Southern blots of digested C. o of these fragments code for the helical region of collagen. They hybridized to the same bands as the intact procollagen clone. The other fragment, which codes for the telopeptide, did not hybridize to any bands but did hybridize visibly to the procollagen clone itself, present as a control in an amount corresponding to a single copy sequence in the worm DNA. About 50 C. es containing procollagen hybridizing sequences have been isolated after screening recombinant phage with the chicken procollagen probe. Screening with the chicken procollagen probe gave about 25 times the frequency of hybridizing plaques as with the Dictyosteleum actin probe. Hybridization to the procollagen probe showed many plaques with intermediate and weak hybridization, unlike hybridization to the actin probe. We do not know the reason why bands and plaques of weak hybridization are seen using the procollagen probe. There may be a region on that clone that is homologous to a repetitive sequence in C. elegans DNA.
[
Worm Breeder's Gazette,
1978]
During the past year we have initiated a study of the DNA of C. elegans with a number of long range objectives in mind. We would like to isolate the DNA from genetically defined regions of the genome in order to construct physical maps to go along with genetic maps. We would like to use isolated fragments of DNA as hybridization probes for studies of transcription. If the size of an isolated restriction fragment differs in two strains of the worm, e.g., Bristol and Bergerac, this size can be used as a phenotype to map the genetic location of the restriction fragment. In this way we hope to locate on the genetic map genes, such as the ribosomal genes, which can be physically isolated but in which mutations have not yet been identified. The size of restriction fragments can also be used as a sensitive method to search for changes in the primary structure of the DNA during development. Here we report our initial progress in these experiments. Isolation of nucleic acids We have found that worms can be completely dissolved and digested by proteinase K in 1% SDS at 65 C, allowing isolation of DNA of high molecular weight and poly-A-containing RNA which is active in an in vitro translation system. Worms, usually frozen in liquid N2 and ground with a mortar before melting, are taken up in .1M tris pH 8.5, . 05M EDTA, .2M NaCl, 1% SDS. (Freezing and grinding is probably not necessary.) Proteinase K (EM Laboratories, Inc.; available from Scientific Products) is added to 200 lambda/ml and the mixture is heated to 65 C for 15' with occasional gentle rocking to mix. During this time the mixture clears almost completely and all worm carcasses disappear. The highly viscous solution is then extracted three times with phenol and once with chloroform-isoamyl alcohol (24:1). DNA may be separated from RNA at this point by precipitating nucleic acids with ethanol and winding out the DNA. We further purify DNA from RNA by digestion with RNase followed by phenol extraction and ethanol precipitation. Unfortunately, worms, particularly adults, contain a particulate material (a polysaccharide?) which copurifies with DNA through organic extractions and ethanol precipitations. This material results in blue DNA solutions, and may be responsible for the indigestibility of some DNA preparations with restriction endonucleases. Much of this material can be removed by spinning the DNA solution at 20,000 rpm for half an hour, and we do this routinely. To obtain C. elegans DNA rigorously free of E. coli DNA, we allow hypochlorited eggs to hatch into M-9 buffer and then purify DNA from the hatched L1's. C. elegans ribosomal DNA DNA coding for 18s and 28s ribosomal RNA (rDNA) can be purified from the bulk of worm DNA as a high density (50% G+C) satellite on cesium chloride gradients (Sulston and Brenner, Genetics 77, 95-104, 1974). The ribosomal genes are tandemly repetitious, containing about 50 copies of each gene. Digestion of this rDNA-containing satellite with restriction endonucleases Bam HI or Sal I gives a single band, the ribosomal unit repeat of 6800 base pairs. The appearance of only one band indicates that the rDNA contains a rather homogeneous repeat, and is the only repetitive DNA in the satellite. This band hybridizes labelled ribosomal sequences at a level 50-100 fold greater than expected for a unique sequence. We have cloned the 6800 base pair Bam fragment thereby providing a probe for hybridization experiments. We have mapped a number of restriction sites within the ribosomal repeat unit. Total worm DNA is digested with the appropriate restriction enzyme(s), run on a 0.7% agarose gel and blotted onto nitrocellulose filter by the technique of Southern. The filter is hybridized to either iodinated 125I-rRNA or to nick-translated 32P- cloned rDNA to identify the restriction fragments containing rDNA. The restriction map is consistent with a homogeneous 6800 base pair unit repeat. Heterogeneous repeat units present in only one copy would not be seen in this analysis, but are currently being looked for. The approximate location of 18s and 28s genes and of spacer regions has been located on the map. Hybridization to Southern blots from heavily loaded gels of digested DNA show a few minor bands, which are presumably fragments from the ends of the tandem repeat, containing some overlap into non-ribosomal DNA. It is also possible that some minor bands are heterogeneous unit repeats. Cloning and characterization of these fragments is in progress. It is striking that the length of the unit repeat is smaller than that found in almost all other eukaryotes. This could be the result of small genes or of very short spacer regions. We have sized the C. elegans large and small rRNAs by electrophoresis of glyoxal-denatured RNA on agarose gels (McMaster and Carmichael, PNAS 74, 4835-4838, 1977) obtaining values of 1700 and 3350 nucleotides, smaller than other eukaryotic rRNAs. In an attempt to locate the ribosomal genes on the genetic map, and to study the inheritance of repetitive DNA, we have compared the restriction cutting patterns of N2 rDNA with those of other strains of C. elegans. Any difference in cutting pattern (most likely due to spacer differences) would be a phenotype, easily mappable. In a comparison of N2 with C. elegans var. Bergerac (J. Brun), rDNA cutting patterns were identical with each of 12 restriction enzymes used. Using several restriction enzymes, the rDNA cutting pattern was also the same with a strain of C. elegans isolated from the wild (D. Russel). rDNA from C. briggsae (B. Zuckerman) did give differences in restriction cutting patterns. The restriction map is similar to that of N2, although the unit repeat is 400 base pairs longer and a few cutting sites are added or deleted. Some fragments appear to be the same in both species. Unfortunately, attempts by us (as well as by Nigon and Dougherty, J. Exp. Zool. 112, 485-503, 1949) to cross C. elegans and C. briggsae have not succeeded. Work at establishing a genetic system for rDNA is continuing. Repeated sequences in C. elegans DNA Sulston and Brenner (Genetics 77, 95-104, 1974) have shown that the DNA of C. elegans contains repetitive components similar to those found in other eukaryotic organisms: namely, inverted repeats, highly repetitive sequences, moderately repetitive sequences, and uniclue sequences. We have undertaken the further characterization of these sequences. So far we have completed initial experiments on the inverted repeat sequences and the moderately repetitive sequences. Inverted repeats. We have studied inverted repeat sequences by electron microscopy and find them to be similar in every way to those found in other eukaryotic organisms. Inverted repeats are visualized by simply melting high molecular weight DNA and spreading it for electron microscopy using the formamide technique of Davis, Simon and Davidson (Methods in Embryology, Vol. XXI, p. 413, 1971). The inverted repeat sequences are then seen as double-stranded stems, or stems with loops at their end, sticking out from the largely single- stranded DNA. Of 37 inverted repeats visualized, those without terminal loops (78%) had stems with a number average length of 250 bp, and those with loops (22%) had stems with a number average length of 340 bp. The number average length of the loops was 800 bp. The inverted repeats appear to be located in clusters in the DNA. Clusters contain a few (3-6) inverted repeats separated by about a thousand base pairs, and clusters are separated from each other by 10 to more than 70 thousand base pairs of DNA containing no inverted repeats. Moderately repetitive sequences. Moderately repetitive sequences are sequences present in the range of 10 to 100 times in the genome. In most eukaryotic organisms about half of such sequences consist of short (300 bp) stretches of repetitive DNA surrounded by unique sequences, and a large fraction of the unique DNA is interspersed in this way, at about one thousand base pair intervals, with moderately repetitive sequences. A few organisms (Drosophila, Chironomous, honey bee, and Achyla--a water mold) lack these short, interspersed repetitive sequences. We have studied the interspersion pattern of repetitive DNA in C. elegans by reassociation kinetics and find that, like Drosophila and the others of the minority group, it appears to lack the highly interspersed component of repetitive DNA. We have compared the rate of reassociation of fragments averaging 300 (120-650) and 2000 (1000- 4000) base pairs in length, using hydroxyapatite binding to assay formation of double strands. DNA of L1's were used after labeling by nick-translation. For shearing, reassociation, and hydroxyapatite binding, the methods of Britten, Graham, and Neufeld (Methods in Enzymology, 29, 363, 1974) have been followed. Seventy-six percent of the 2000 base pair fragments reannealed at a rate expected for unique fragments of that length. This represents only a slight increase over the fraction of the 300 base pair fragments which carry repetitive DNA, an increase from 20% to 24%. This result is consistent with a lack of highly interspersed repetitive DNA. We are presently analyzing the length of moderately repetitive sequences by electron microscopy to determine whether any short repetitive sequences are present at all. Studies on cloned fragments of C. elegans DNA We have constructed a small clone bank of C. elegans restriction fragments. We have used the Bam restriction endonuclease and have inserted the fragments into the pBR313 driver plasmid. Recombinant DNA work with C. elegans DNA is at the P2-EK1 level. We will be happy to share recombinant plasmids. We are using the cloned fragments as hybridization probes to study restriction fragments in worm DNA. A restriction digest of whole- genome DNA is fractionated on an agarose gel and transferred to a millipore filter (a 'Southern transfer'). A plasmid containing a particular cloned fragment is then labeled by nick-translation and hybridized to the filter to reveal the fragments in the whole digest which carry sequences homologous to those of the cloned fragment. We have been analyzing the patterns produced in this way to answer a number of questions: 1. Are the patterns consistent with the arrangement of repetitive DNA determined by COT analysis; that is, do most fragments consist solely of unique sequences? 2. Are there any differences in the patterns given by germ-line and somatic-line DNAs? 3. Are there any differences in the patterns given by Bristol and Bergerac DNAs? These could be used for mapping. Are there any differences between C. elegans and C. briggsae patterns? 4. Can differences in these patterns be used to find cloned fragments that come from genetically defined regions, for example, from regions covered by deletions? By hybridizing 0.1 g of a plasmid nick-translated to more than 10+E7cpm/ g to a filter carrying a digest of a few micrograms of worm DNA we can detect unique fragments after an overnight exposure. We use flashed film and intensifying screens and expose the film at -70 C. We have found that hybridizations at low temperature (e.g., 32 C) in 50% formamide and without Denhardt's solution are convenient and work very well. Thirteen recombinant plasmids (with inserts ranging in size from 1, 000 to 18,000 base pairs) have been hybridized to filters carrying digests of DNA from N2 L4 hermaphrodites, N2 L1 hermaphrodites, Bergerac L1 hermaphrodites, and C. briggsae (mixed population). All hybridize to a fragment in N2 DNA equal in size to the cloned insert they carry, indicating that no rearrangements have taken place during cloning. Nine plasmids hybridize to several (up to 10) additional bands. Even most inserts of less than 2000 base pairs (5 out of 8) hybridize to more than one band. From the COT analysis described earlier we would expect 76% of such fragments to consist entirely of unique DNA. Whether these figures are inconsistent, and if so, why, remains to be seen. We have used L4 hermaphrodites as a source of 'germ line' DNA in these experiments. By comparing DNA from them to DNA from newly hatched L1's, which lack a gonad, we can search for restriction fragments present in the germ line but absent from the somatic line. No such fragments have been found; so far the L1 and L4 patterns are identical. We have also started to use DNA isolated from sperm nuclei (a gift from Michael Klass), which will allow a much more rigorous comparison of germ and somatic line sequences. (In addition we are hoping that a comparison of sperm and hermaphrodite DNAs will allow, by examining the relative intensities of bands, identification of fragments from the X-chromosome.) Comparison of the bands in Bergerac and Bristol DNA's shows that these DNAs are not identical. Five Bristol bands (including two of the cloned inserts) appear to have a different size in Bergerac; that is, they are missing in the Bergerac pattern and one new band is present. We would like to find out whether these differences are due to single base changes or to rearrangements. This degree of difference between these strains suggests that genetic mapping by restriction fragments is feasible. Comparison of the C. elegans patterns with those of C. briggsae shows (to our surprise) that these DNAs are highly diverged. None of the 13 cloned fragments is present unaltered in the briggsae genome, and in fact 9 hybridize to no fragment whatsoever in briggsae DNA. Since we expect (but have not checked) that the proteins of these ( almost indistinguishable) worms would be very similar, this raises the possibility that DNA sequences present in both species are coding sequences.
[
Worm Breeder's Gazette,
1982]
We thought that
unc-92, a dominant unc with disorganized muscle and a high reversion frequency, might be an actin gene because of its phenotype and because of its map position (Newsletter 6, 23 (1981)). Using a Bergerac strain/Bristol strain DNA polymorphism adjacent to the cluster of actin genes I, II and III, we were able to locate the actin cluster between
unc-23 and
sma-1. The arrangement of actin genes I, II and III within a 12 kb stretch of DNA suggested mechanisms for the high
unc-92 reversion frequency, including gene conversion between genes and nonhomologous recombination. Nonhomologous recombination should reduce three genes to two which would be easy to detect by Southern blot analysis. We therefore obtained from Bob Waterston six revertants of the St15 allele of
unc-92; three revertants arose spontaneously and three were obtained after an EMS mutagenesis. We isolated DNA from each revertant strain and analyzed it by Southern blot analysis. Five of the revertants showed a pattern identical to that of both St15 and N2 when probed for actin sequences, but one revertant, RW2246, which derived from the EMS mutagenesis, showed an anomalous pattern. Further restriction mapping of RW2246 by Southern blot analysis revealed that this revertant contained a 5kb deletion that fused the 5' portion of gene II to the 3' portion of gene III. The fusion could have been the result of recombination between genes II and III, deleting the
unc-92 lesion and creating a new hybrid gene. We believe that the revertant phenotype resulted from this fusion. Because most of the differences between actin genes occur at their 5' ends, the fusion leaves the revertant with the equivalent of only genes I and II. Despite the lack of gene III, the revertant appears wild type because genes I and III appear thus far to be identical by restriction endonuclease and sequence analyses. We therefore believe that
unc-92 is the actin gene cluster, although final proof awaits comparison of the actin DNA sequences of N2 and St15.