Worm Breeder's Gazette 3(2): 34

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Studies on the DNA of C. elegans

S. Emmons, J. Files, B. Rosenzweig

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 
Isolation of nucleic 
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 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 
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 
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 
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