The linkage mapping of cloned restriction fragment length differences in Caenorabditis elegans

Summary The genomic DNA of two closely related strains of the nematode, Caenorhabditis elegans, Bristol (N2), and Bergerac (Bo), has different restriction endonuclease sites (Emmons et al. 1979). Since these two strains interbreed, it is possible to regard the restriction fragment length differences (RFLDs) as mutant variants. The N2 and Bo pattern can be segregated and mapped using clasical genetic techniques.Utilizing a number of genetic markers existing in the N2 strain, we have constructed hybrid populations homozygous for either Bristol or Bergerac over a given chromosomal region with random Bristol-Bergerac composition for the remainder of the genome. Genomic restriction digests from these hybrid populations were probed with random cloned fragments of Bristol DNA. In this way, fragments were mapped to genetically well characterized regions of the C. elegans genome. 27 probes which hybridize to a total of 310 Kb of DNA were found to exhibit six restriction fragment differences. Four of these differences have been mapped, providing probes for four different genomic regions. We have combined classical genetics and recombinant DNA technology to construct linkage maps of cloned DNA fragments using restriction fragment length differences. We are pursuing this approach in order to advance the knowledge of the genetic organization of C. elegans and to provide a means of cloning genes in an organism which provides an experimental model for the study of many biological systems. It is hoped that this approach will also provide a practical solution to some difficult problems in nematode strain identification. Furthermore, the characterization of the families of transposable elements responsible for generating many of the RFLDs will undoubtedly contribute to the understanding of the biological significance of these elements.     

The Tc5 Family of Transposable Elements in Caenorhabditis Elegans

We have identified Tc5, a new family of transposable genetic elements in the nematode Caenorhabditis elegans. All wild-type varieties of C. elegans that we examined contain 4-6 copies of Tc5 per haploid genome, but we did not observe transposition or excision of Tc5 in these strains. Tc5 is active, however, in the mut-2 mutant strain TR679. Of 60 spontaneous unc-22 mutations isolated from strain TR679, three were caused by insertion of Tc5. All three Tc5-induced mutations are unstable; revertants result from precise or nearly precise excision of Tc5. Individual Tc5 elements are similar to each other in size and structure. The 3.2-kb element is bounded by inverted terminal repeats of nearly 500 bp. Eight of the ten terminal nucleotides of Tc5 are identical to the corresponding nucleotides of Tc4. Further, both elements recognize the same target site for insertion (CTNAG) and both cause duplication of the central TNA trinucleotide upon insertion. Other than these similarities to Tc4, Tc5 is unrelated to the three other transposon families (Tc1, Tc3 and Tc4) that transpose and excise at high frequency in mut-2 mutant strains. Mechanisms are discussed by which four apparently unrelated transposon families are all affected by the same mut-2 mutation.     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

Somatic excision of transposable element Tc1 from the Bristol genome of Caenorhabditis elegans.

We investigated the ability of the transposable element Tc1 to excise from the genome of the nematode Caenorhabditis elegans var. Bristol N2. Our results show that in the standard lab strain (Bristol), Tc1 excision occurred at a high frequency, comparable to that seen in the closely related Bergerac strain BO. We examined excision in the following way. We used a unique sequence flanking probe (pCeh29) to investigate the excision of Tc1s situated in the same location in both strains. Evidence of high-frequency excision from the genomes of both strains was observed. The Tc1s used in the first approach, although present in the same location in both genomes, were not known to be identical. Thus, a second approach was taken, which involved the genetic manipulation of a BO variant, Tc1(Hin). The ability of this BO Tc1(Hin) to excise was retained after its introduction into the N2 genome. Thus, we conclude that excision of Tc1 from the Bristol genome occurs at a high frequency and is comparable to that of Tc1 excision from the Bergerac genome. We showed that many Tc1 elements in N2 were apparently functionally intact and were capable of somatic excision. Even so, N2 Tc1s were prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tc1 elements have the ability to transpose but that transposition is heavily repressed in the gonadal tissue.     

Analysis of the Caenorhabditis Elegans Axonal Guidance and Outgrowth Gene Unc-33

Mutations in the unc-33 gene of the nematode Caenorhabditis elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. We have cloned unc-33 by tagging the gene with the transposable element Tc4. Three unc-33 messages, which are transcribed from a genomic region of at least 10 kb, were identified and characterized. The three messages have common 3' ends and identical reading frames. The largest (3.8-kb) message consists of the 22-nucleotide trans-spliced leader SL1 and 10 exons (I-X); the intermediate-size (3.3-kb) message begins with SL1 spliced to the 5' end of exon V and includes exons V-X; and the smallest (2.8-kb) message begins within exon VII and also includes exons VIII-X. A gamma-ray-induced deletion mutation situated within exon VIII reduces the sizes of all three messages by 0.5 kb. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein. A Tc4 insertion in exon VII leads to alterations in splicing that result in three approximately wild-type-size messages: the Tc4 sequence and 28 additional nucleotides are spliced out of the two larger messages; the Tc4 sequence is trans-spliced off the smallest message such that SL1 is added 13 nucleotides upstream of the normal 5' end of the smallest message.     

Evidence for a transposon in caenorhabditis elegans

The C. elegans genome contains a 1.7 kb repeated DNA sequence (Tc1) that is present in different numbers in various strains. In strain Bristol and 10 other strains analyzed, there are 20 ± 5 copies of Tc1, and these are located at a nearly constant set of sites in the DNA. In Bergerac, however, there are 200 ± 50 interspersed copies of Tc1 that have arisen by insertion of Tc1 elements into new genomic sites. The interspersed copies of Tc1 have a conserved, nonpermuted structure. The structure of genomic Tc1 elements was analyzed by the cloning of a single Tc1 element from Bergerac and the comparison of its structure with homologous genomic sequences in Bristol and Bergerac. Tc1 elements at three sites analyzed in Bergerac undergo apparently precise excision from their points of insertion at high frequency.     

Spontaneous Unstable UNC-22 IV Mutations in C. ELEGANS Var. Bergerac

This paper describes a mutator system in the nematode Caenorhabditis elegans var. Bergerac for the gene unc-22. Of nine C. elegans and two C. briggsae strains tested only the Bergerac BO strain yielded mutant animals at a high frequency and the unc-22 IV gene is a preferred mutational target. The forward spontaneous mutation frequency at the unc-22 locus in Bergerac BO is about 1 x 10^-4 , and most of these spontaneous unc-22 mutations revert at frequencies between 2 x 10^-3 and 2 x 10 ^-4. Both the forward mutation frequency and the reversion frequency are sensitive to genetic background. Spontaneous unc-22 mutations derived in a Bergerac background and placed in a primarily Bristol background revert at frequencies of <10^-6. When reintroduced into a Bergerac/Bristol hybrid background the mutations once again become unstable.

The mutator activity could not be localized to a discrete site in the Bergerac genome. Nor did mutator activity require the Bergerac unc-22 gene as a target since the Bristol unc-22 homolog placed in a Bergerac background also showed high mutation frequency. Intragenic mapping of two spontaneous unc-22 alleles, st136 and st137, place both mutations in the central region of the known unc-22 map. However, these mutations probably recombine with one another, suggesting that the unstable mutations can occur in more than one site in unc-22. Examination of the phenotypic effect of these mutations on muscle structure indicates that they are less severe in their effect than a known amber allele. We suggest that this mutator system is polygenic and dispersed over the nematode genome and could represent activity of the transposable element Tc1.

     

Germline excision of the transposable element Tc1 in C. elegans.

We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.     

Structural analysis of Tc1 elements in Caenorhabditis elegans var. Bristol (strain N2)

The transposable element Tc1 in the genome of Caenorhabditis elegans var. Bristol strain N2 is very stable. In order to investigate possible causes of Tc1 immobility in this strain 17 individual isolates have been cloned and characterized with regard to their structure and genomic environment. Ten of 16 elements examined had identical restriction maps, and at least 1 of these (#7) showed a high level of somatic excision. Two of the elements had altered restriction sites, 2 had different internal deletions of about 700 bp, 1 had an 89-bp terminal deletion, and 1 a 54-bp insertion. When DNA sequences flanking the N2 Tc1 elements were used as probes in genomic hybridizations, it was found that most N2 elements are located in regions of repetitive DNA. Furthermore when hybridizations to DNA from N2 and var. Bergerac strain B0 were performed, a major band of the same size was observed in both strains. Two flanking sequences identified strain polymorphic sites hP2(IV) and hP3(IV). In at least one of these cases, a rearranged Tc1 was present in the B0 strain at the same location. The fact that all or most of the Tc1 elements are in the same location in N2 and B0 adds support to the hypothesis that the high copy number B0 strain arose from amplification of Tc1 copies in a N2-like strain. The N2 Tc1 elements are highly conserved; however, intact elements had fewer nucleotide changes than the rearranged elements. These results may indicate that the intact Tc1 elements in N2 are functionally active and subject to selective pressure.     

Molecular analysis of the rat MHC. II. Isolation of genes that map to the RT1.E-grc region

An initial mapping analysis of growth and reproduction complex (grc) and grc+ genomic DNA identified several restriction fragment length polymorphisms specific for the grc region of the MHC. To analyze further the genomic organization and structure of the grc, a cosmid library was constructed from a grc+-bearing strain (R21). One cosmid cluster, encompassing 41.4 kb of DNA, contained four, or possibly five, class I genes that mapped to the RT1.E-grc region Two unique non-class I fragments were isolated from certain cosmids within this cluster. These fragments were hybridized to genomic DNA derived from five rat strains (BIL/2, R18, R21, R22, and BIL/1), and the results showed that grc-bearing rats have a deletion of at least 3.1 kb of DNA in the region immediately adjacent to the MHC. The loss of the genes in this region is probably the cause of the growth and reproductive defects in these animals and probably also of their increased susceptibility to chemical carcinogens.     

The Unc-22(iv) Region of Caenorhabditis Elegans: Genetic Analysis of Lethal Mutations

The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.     

Molecular and Genetic Analysis of Unc-7, a Caenorhabditis Elegans Gene Required for Coordinated Locomotion

Mutations in the Caenorhabditis elegans gene unc-7 confer an uncoordinated phenotype. Wild-type animals trace smooth, sinuous waves as they move; unc-7 mutants make irregular bends or kinks along their bodies, particularly when they move forward. The unc-7 locus has also been implicated in the nematode's response to volatile anesthetics. We have cloned unc-7 by transposon tagging: an unc-7 mutation was correlated with the insertion of the transposon Tc1, and reversion of the mutant phenotype was correlated with loss of the Tc1 element. We have physically mapped the region flanking the sites of Tc1 insertion and identified DNA rearrangements corresponding to eight additional unc-7 alleles. Northern analysis indicates that a 2.7-kb unc-7 message is present in all developmental stages but is most abundant in L1-L3 larvae. The 5' end of the message contains a trans-spliced leader SL1. An 18-kb intron is located upstream of the predicted translational start site of the gene, and DNA breakpoints of four gamma-ray-induced alleles were located within this intron. We determined the sequence of a cDNA corresponding to the unc-7 message. The message may encode a 60-kd protein whose amino acid sequence is unrelated to any other available protein sequence; a transmembrane location for the unc-7 protein is predicted. We predict from our analysis of unc-7 genetic mosaics that the unc-7 gene product is not required in muscle cells for wild-type coordination but is probably required in motor neurons (although a hypodermal role has not been excluded). We speculate that unc-7 may be involved in the function of neuronal ion channels.     

Isolation and mapping of DNA probes within the linkage group I gene cluster of Caenorhabditis elegans

We have isolated probes for DNA polymorphisms across the linkage group I gene cluster in Caenorhabditis elegans, using Tc1-linkage selection. The probes detect strain polymorphism between the wild-type strains of var. Bristol and var. Bergerac. As a result of mapping the sites hP4, hP5, hP6, hP7, hP9, and sPl, more than 1000 kilobases (kb) of cloned cosmid DNA has been positioned on the genetic map. We found there is more DNA per map unit in the center of the gene cluster than expected on the basis of the genomic average. Furthermore, the amount is not constant across the entire region but reaches a peak in the hP9 unc-13 interval. To find the coding regions, we examined DNA cross-homology between two species, Caenorhabditis elegans and Caenorhabditis briggsae. Approximately one-third of the DNA in the hP5 hP9 interval was examined for coding regions and 21 sequences were identified within 318 kb of DNA.     

Detection of mutations and DNA polymorphisms using whole genome Southern Cross hybridization.

We report a general method for the detection of restriction fragment length alterations associated with mutations or polymorphisms using whole genomic DNA rather than specific cloned DNA probes. We utilized a modified Southern Cross hybridization to display the hybridization pattern of all size-separated restriction fragments from wild-type Caenorhabditis elegans to all the corresponding fragments in a particular mutant strain and in a distinct C. elegans variety. In this analysis, almost all homologous restriction fragments are the same size in both strains and result in an intense diagonal of hybridization, whereas homologous fragments that differ in size between the two strains generate an off-diagonal spot. To attenuate the contribution of repeated sequences in the genome to spurious off-diagonal spots, restriction fragments from each genome were partially resected with a 3' or 5' exonuclease and not denatured, so that only the DNA sequences at the ends of these fragments could hybridize. Off-diagonal hybridization spots were detected at the expected locations when genomic DNA from wild-type was compared to an unc-54 mutant strain containing a 1.5 kb deletion or to a C. elegans variety that contains dispersed transposon insertions. We suggest that this modified Southern Cross hybridization technique could be used to identify restriction fragment length alterations associated with mutations or genome rearrangements in organisms with DNA complexities as large as 10(8) base pairs and, using rare-cutting enzymes and pulse-field gel electrophoresis, perhaps as large as mammalian genomes. This information could be used to clone fragments associated with such DNA alterations.     

Mutations in the unc-41 Gene Cause Elevation of Acetylcholine Levels

Abstract: Mutations in the Caenorhabditis elegans unc-41 gene result in an allele-dependent elevation of acetylcholine content. Eight recessive alleles ( cn252, e268, e399, e650, e1175, e1199, e1294, and e870 ) lead to phenotypes including uncoordinated locomotion, slow growth, a small mature body, and resistance to the acetylcholinesterase inhibitors as well as the elevation of acetylcholine content. The remaining two alleles, e554 and e1162 , exhibit normal acetylcholine levels but display the short-body phenotype in a semidominant way. To determine the localization of the elevated acetylcholine content, a method for the isolation of synaptic vesicles from C. elegans was established. The elevation of acetylcholine content in the unc-41 mutants is accompanied by the accumulation of synaptic vesicles. We propose that at least one function of the unc-41 gene relates to the release of neurotransmitters.     

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.