[
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.
[
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,
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,
1987]
We determined the DNA sequences of several collagen genes with different expression patterns during development and compared them to the previously sequenced genes
col-1 and
col-2. The genes chosen for study were
col-1 and
col-14 which are expressed at varying levels throughout development,
col-2 and
col-6 which are dauer-specific and
col-7, e expressed primarily in animals molting into adults. Each gene is 1.0 to 1.2 kb in length and includes one or two short introns at variable positions. The presumptive promoter regions contain the expected eukaryotic TATA and CAAT sequences. The sequence TAT CTTTCTCTY TTCTTYCT (Y=C or T) is present 30 bp and 74 bp upstream of the CAAT box in
col-2 and
col-6, respectively. The sequence AAATTT YAYCAATRT TTATT AATT is present 203 and 183 bp upstream of the presumptive CAAT boxes in
col-7 and
col-19 ( R=A or G; the relevant region of
col-8 was not sequenced). The correlation between the presence of these sequences and the similar expression profiles of the relevant genes suggests that these sequences may be involved in the developmental regulation of the genes. The domain structure of the collagen polypeptides is similar to that determined for
col-1 and
col-2: each polypeptide contains two main triple-helix forming (Gly-X-Y)n domains, one of 30-33 amino acids, and the other of 127-132 amino acids. The latter domain is interrupted by short (2-8 amino acids) non-Gly-X-Y segments in each polypeptide. Sets of cysteine residues flank the (Gly-X-Y)n domains in all of the polypeptides. The genes can be placed into three families based upon structural features (overall protein length and organization of (Gly-X- Y)n domains), positions of cysteine residues and amino acid sequence homologies. The amino acid sequence homologies are most evident in the non-Gly-X-Y domains. As an example, the C-terminal tail sequences are shown below. Col-1 and
col-2 comprise one family,
col-6 and col- 14 comprise a second family and
col-8 and
col-19, with the less homologous
col-7, comprise the third family. Members of a family can be coordinately regulated as in the case of
col-8, ave different expression patterns as in the cases of
col-1 and
col-2 or
col-6 and
col-14.The codon usage in all of the genes is highly asymmetrical, with adenine appearing in the third position of 85% of the Gly codons and 93% of the Pro codons.
[
Worm Breeder's Gazette,
1982]
We have isolated DNA from most of the new wild isolates of C. elegans described elsewhere in this issue by Dick Russell and coworkers and analyzed the pattern of bands produced when a cloned copy of the transposon Tc1 is hybridized to a restriction digest on a Southern. From the fractionated restriction digests we have found that all these strains have a ribosomal repeat length identical to Bristol. The hybridization with Tc1 gave two interesting findings. One is that all the wild strains have a series of bands similar in number to Bristol and clearly different from the smear in Bergerac. Thus Bergerac may be an unusual case and represent aberrant regulation of the transposon. The second finding is that some differences in the pattern of bands are seen among these strains. The same result was reported earlier by Rosenzweig, Liao and Hirsh, Newsletter Vol. 7, no. 1, p.51. We assume without direct evidence that these differences are due to transposition of Tc1 because we know that transpositions have occurred more frequently than point mutations during the divergence of Bristol and Bergerac. Our Southerns so far visualize as bands a minority of the hybridizing material, most of which remains in large, unfractionated fragments at the top of the gel, and therefore our analysis must be considered preliminary. Nevertheless, based on the fragments we do see, we find the strains falling into the following categories (with the number of bands differing relative to Bristol (N2) shown in parentheses) : [See Figure 1] It seems very surprising on the one hand that worms isolated in California in the 1970's should be so similar to a worm isolated in England in the 1940's, and on the other hand that worms isolated from a single garden (the Ga series) should differ as much from each other as they do from the older English isolate. The first fact might reflect a greater degree of world-wide mixing of worms (wind, water, birds' feet?) than we had naively supposed. The second might result from reproduction by selfing, which suppresses genetic exchange within a population.
[
Worm Breeder's Gazette,
1989]
unc-4 appears to define the wiring diagram for a specific portion of the ventral nerve cord. In
unc-4(
e120), most VAn motor neurons assume a pattern of presynaptic connections normally reserved for the VBn motor neuron class; VAn's receive input from AVB and PVC interneurons as opposed to the normal sets of connections with AVA, AVD and AVE which are absent in
e120 (White et al., WBG 9,
p81, 1985). We have now shown that
unc-4 may correspond to yet another homeobox locus in C. elegans.A spontaneous allele,
unc-4(
wd1), was isolated from the mutator strain TR679 in a general screen for movement-defective mutants. Blots of recombinants with flanking markers
rol-6 and
bli-1 identified two closely linked TC1-containing EcoRI fragments; wdP1 (2. 5 kb) is 0.12 m.u to the right and wdP3 (3.2 kb) is 0.1 m.u to the left. A phage clone overlapping wdP1 was fingerprinted in Cambridge and maps to a large contig near
unc-4 on chromosome II. wdP3 includes repetitive DNA and a corresponding phage clone to mark the left side boundary is apparently not represented in our genomic library. The identification of a homeobox sequence in the region, however, suggested a likely location for
unc-4. Cosmid C07E2 overlaps,
ceh-4, one of 50 different homeobox loci that hybridize with a degenerate oligonuclcotidc probe (T. Burglun and G. Ruvkun, WBG 10,
p35, 1988). We probed Southern blots of DNA isolated from 15 independent
unc-4 alleles with random primer labeled C07E2. Alterations were detected in five of these alleles:
e2314,
e2320,
e2308 (from M. Shen),
e887 and wd-1. All of these mutations affect a 1.8 kb EcoRI fragment (see below). The
e2320 mutation deletes 200 bp from the 1.8 kb fragment. In wd-1, the 1.8 kb as well as several other EcoRI fragments are absent suggesting a large deletion (~25kb). The alterations observed in
e2314,
e2308, and
e887 have not been reconstructed but all of these mutations delete the 1.8 kb EcoRI fragment. A restriction map of the region indicates that a 1.05 kb EcoRI fragment containing the homeobox 'third helix' consensus sequence (.WFQNRR.K.R) is adjacent to the region that is altered in
unc-4 mutants. We are now searching for a cDNA to determine if in fact the putative homeodomain corresponds to portion of the
unc-4 coding sequence. [See Figure 1]