[
Worm Breeder's Gazette,
1982]
A hermaphroditic nematode strain isolated from the soil (by A. F.) in the State of Gujarat, India, has been identified as Caenorhabditis briggsae by genetic and morphological criteria. The Dougherty strain of C. briggsae, which has been cultured axenically in various laboratories since 1954, was obtained by the CGC from Dr. Bert Zuckerman. No other sub-lines of C. briggsae are known to exist (see Friedman, Platzer and Eby, 1977, J. Nematol. 9:198). A male stock of the Gujarat strain was established and used for mating tests, genetic analysis and comparisons of male copulatory bursa morphology together with N2 males and heat-shock-generated Zuckerman males. ( Historically, the number and arrangement of bursal rays has been the basis for species classification in this genus.) The Gujarat males mate very well with the Zuckerman strain and not at all with C. elegans N2. Conversely, N2 males fail to mate with either Gujarat or Zuckerman hermaphrodites. Examination of bursal ray patterns confirms the conclusion that the Gujarat strain is C. briggsae. In general, the behavior, appearance, and growth properties of the Gujarat strain are very similar to C. elegans strain N2. However, both of the C. briggsae strains grow and reproduce well at 27.5 C while N2 does not. The Gujarat generation time from egg to egg is about 37 hrs. at 27.5 C, and about 56 hrs. at 20 C. The Zuckerman C. briggsae strain differs markedly from both Gujarat and N2 in many respects. The Zuckerman strain is variably uncoordinated, it exhibits chemotaxis defects, it is dauer-defective the hermaphrodites produce relatively small broods (140 + 18 is the best at 22.5 C in comparison with 238 + 25 of Gujaratat the same temperature), spontaneous males will not mate but instead crawl off the plate, and the male bursa is abnormally small with stunted rays. Bursa size and ray length of Gujarat males are the same as N2, but 25% greater than Zuckerman males. Gujarat/Zuckerman hybrid males possess a bursa indistinguishable from Gujarat itself, so we conclude that this Zuckerman trait is autosomal and recessive. However, the hybrid males are uncoordinated, non-chemotactic, and dauer-defective. The dauer-defective phenotype can be scored as a failure to form dauer larvae in response to the Caenorhabditis 'crowding' pheromone (Golden and Riddle, 1981, C. elegans Meeting Abstracts). These traits are all X-linked, recessive, and may represent pleiotropic effects of a single mutation. An attempt was made to induce revertants of the Zuckerman dauer-defective trait with EMS, but no revertants were found in a screen of almost 10+E5 mutagenized chromosomes. This leaves open the possibility that the X-linked defect may be a non-revertible, multi-site mutation of some sort. What does all this mean? Perhaps not much, but we believe this may be a particularly grandiose example of evolution (or de-evolution) in the laboratory. The Dougherty strain of C. briggsae was reported in 1969 to form dauer larvae (Yarwood and Hansen, 1969, J. Nematol. 1:184), but the strain we have today forms dauers at very low frequency. We are tempted to speculate that prolonged liquid axenic cultivation may have actually favored the type of genetic variation in sensory behavior, movement and dauer formation that we have observed in the Zuckerman strain. Interestingly, the Zuckerman strain does not exhibit uncoordinated movement in liquid, but it seems to thrash about more slowly than N2 or Gujarat. Slower movement also might be an advantage in liquid suspension culture. Does cultivation on agar plates spread with E. coli also select particular types of variation from 'true wild-type'? Well, our N2 stock is not noticeably different in growth or behavior from the recently-isolated Gujarat C. briggsae strain, with one interesting exception. The exception involves sensitivity to the dauer larva pheromone. All three strains produce the Caenorhabditis specific 'crowding' pheromone. While the Zuckerman strain does not respond to the pheromone (it is dauer-defective), the Gujarat strain responds to even lower concentrations of pheromone than does N2. Presumably as a consequence of its pheromone sensitivity, the Gujarat strain actually forms some dauers prior to starvation on NGM plates. If N2 had such a characteristic when it was first cultured on E. coli, it almost certainly would have been at a selective disadvantage to spontaneous genetic variants less sensitive to the pheromone.
[
Worm Breeder's Gazette,
1983]
Using techniques developed by Judith Kimble (1), we have been microinjecting supercoiled plasmid DNA into the gonad distal arm of young adult hermaphrodites. To detect the inheritance of exogenous DNA, progeny of the injected worm are grown for approximately three generations and then harvested. Their DNA is extracted and tested for the presence of the injected sequences by hybridization to the plasmid DNA. To date 11 of 119 injections have shown evidence for the inheritance of foreign DNA. The organization of foreign DNA in worm transformants has been examined by restriction analysis. The exogenous DNA is present as high molecular weight DNA at high copy number (20-100 copies per transformed worm) and arranged predominantly as a head-to-tail tandem array. In one transformant injected with YRp17-Cell0, a plasmid containing sequences with worm homology, the exogenous DNA appears to have integrated into the worm genome by homologous recombination. Two independent transformants containing DNA with no extensive homology to the worm have been tested for the stability of foreign sequences during propagation. The exogenous DNA is lost in these transformants at a fairly high rate. For a majority of subclones of a transformant, only 50% of the progeny in the next generation still contain foreign DNA. By restriction analysis, the foreign DNA in all the subclones of a single transformant appears to be identical high molecular weight concatamers. Thus, the exogenous DNA appears to segregate as a single unit that is not undergoing rearrangements at a high frequency. The high instability of this unit suggests that either the foreign DNA imparts a deleterious phenotype to the worms, or the concatamer is extrachromosomal and is being lost during mitosis or meiosis. We are presently investigating these alternatives. Recently several subclones have shown high stabilities during propagation. We are now trying to determine the cause of this increase in stability. To assess the frequency of homologous recombination of injected plasmid sequences into the worm genome, we have injected both supercoiled and linear plasmids containing portions of the
unc-54 gene. Homologous integration of these DNA sequences should result in the production of an
unc-54 mutation by disruption of the endogenous gene. We have yet to demonstrate that exogenous DNA can be expressed. Plasmids containing the wild-type
unc-54 gene or the amber-suppressing
sup-7 allele have been injected to assess complementation of
unc-54 mutants or suppression of amber mutants at several loci. In addition, we are constructing hybrid genes that could provide a selection or a visible screen for transformed worms. We have recently injected a - glucuronidase negative worm with a vector carrying the E. coli - glucuronidase gene fused to the
col-1 5'- and 3'- sequences. Transformants containing this vector are being tested for - glucuronidase activity. Transformants containing a similar vector carrying the bacterial structural gene for neomycin phosphotransferase are being tested for resistance to the drug G418. Dr. Hans-Borje Jansson, Univ. of Lund, Sweden joined my program in July, 1983. He is supported by a Swedish National Science Foundation Fellowship and a Fulbirght Award. Concurrently Dr. A. Jeyaprakash of India joined us. We are working on trying to characterize surface carbohydrates on C. elegans and attempting to intervene in chemotactic behavior using various experimental manipulations. Bert M. Zuckerman, Professor
[
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.