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Previous ArticleNext Article

Mapping a Telomere Using the Translocation eT1(III;V) in Caenorhabditis elegans

K. A. Adames, Jocelyn Gawne, Chantal Wicky, Fritz Müller and Ann M. Rose
Genetics November 1, 1998 vol. 150 no. 3 1059-1066
K. A. Adames
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Jocelyn Gawne
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Chantal Wicky
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Fritz Müller
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Ann M. Rose
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Abstract

In Caenorhabditis elegans, individuals heterozygous for a reciprocal translocation produce reduced numbers of viable progeny. The proposed explanation is that the segregational pattern generates aneuploid progeny. In this article, we have examined the genotype of arrested embryonic classes. Using appropriate primers in PCR amplifications, we identified one class of arrested embryo, which could be readily recognized by its distinctive spot phenotype. The corresponding aneuploid genotype was expected to be lacking the left portion of chromosome V, from the eT1 breakpoint to the left (unc-60) end. The phenotype of the homozygotes lacking this DNA was a stage 2 embryonic arrest with a dark spot coinciding with the location in wild-type embryos of birefringent gut granules. Unlike induced events, this deletion results from meiotic segregation patterns, eliminating complexity associated with unknown material that may have been added to the end of a broken chromosome. We have used the arrested embryos, lacking chromosome V left sequences, to map a telomere probe. Unique sequences adjacent to the telomeric repeats in the clone cTel3 were missing in the arrested spot embryo. The result was confirmed by examining aneuploid segregants from a second translocation, hT1(I;V). Thus, we concluded that the telomere represented by clone cTel3 maps to the left end of chromosome V. In this analysis, we have shown that reciprocal translocations can be used to generate segregational aneuploids. These aneuploids are deleted for terminal sequences at the noncrossover ends of the C. elegans autosomes.

THE cloning of the telomeres and their adjacent sequences has provided probes for the ends of the Caenorhabditis elegans chromosomes (Wickyet al. 1996). Wicky et al. (1996) were able to assign one telomere to the right end of chromosome I (cTel29) and one to the left end of the X chromosome (cTel7). In the first case, the identification of ribosomal DNA sequences in the clone cTel29 provided the clue that this telomere originated from DNA adjacent to the rDNA gene cluster on chromosome I. In the second case, characterization of a terminal deletion of the X chromosome showed the absence of cTel7 sequences. However, terminal deletions for the other chromosome ends were not available. Further attempts to map clones by hybridization to filters containing ordered arrays of cosmids or yeast artificial chromosomes (YACs) were not successful because the chromosome ends were absent from the available libraries. In this article, we show that aneuploid embryos, arising from the segregation of translocated chromosomes, provide a source of tip deletions useful for mapping loci at the ends of the C. elegans chromosomes.

In C. elegans, aneuploid progeny have been predicted to arise in the segregants of individuals heterozygous for a reciprocal translocation (Herman 1978; Rosenbluth and Baillie 1981). In the case of eT1(III;V), which is viable as a homozygote, Rosenbluth and Baillie (1981) predicted the production of specific inviable aneuploids in the progeny of heterozygotes carrying the reciprocal translocation. Among the expected progeny are homozygous deletions for the left half of chromosome V, and for the right half of chromosome III. These deletions differ from those created by a mutagen in that they are generated by segregational patterns. In the case of mutagen-induced tip deletions, the exact sequence of the DNA at the end of the deleted chromosome is rarely known. In the case of translocation segregants, on the other hand, there simply is no DNA present in the missing region, thus facilitating the interpretation of the results.

eT1 is a stable, well-characterized reciprocal translocation, which has the left portion of chromosome III joined to the left portion of chromosome V (Rosenbluth and Baillie 1981). This half translocation recombines and disjoins from the normal chromosome III in heterozygotes and has been designated eT1(III). The right portion of chromosome III is joined to the right portion of chromosome V forming eT1(V). Each of the half-translocations consistently recombines with, and disjoins from, only one of the two normal sequence chromosomes. That is, eT1(III) always crosses over and disjoins from the normal chromosome III; whereas eT1(V) always crosses over and disjoins from the normal chromosome V. At the same time, crossing over is essentially eliminated for the right portion of chromosome III and the left portion of chromosome V. The fact that crossing over is strongly suppressed along the entire length of these translocated segments suggests that the absence of crossing over may result from a failure of these segments to pair with their homologs. Predictions of expected progeny based on this pairing configuration agree with the data observed by Rosenbluth and Baillie (1981). Included in the predicted segregants are progeny homozygous for deletions of the crossover suppressed regions. However, before this study molecular proof of the existence of these deletion homozygotes was lacking.

Heterozygotes for eT1 are effective balancers for large segments of chromosome III (right) and chromosome V (left), where crossing over is essentially eliminated. The eT1 translocation has been used extensively for the recovery, maintenance and analysis of unconditional recessive lethal mutations (Rosenbluth et al. 1983, 1985, 1988; Johnsen and Baillie 1988, 1991; reviewed in Edgleyet al. 1995). However, the developmental arrest stages for each of the progeny classes from eT1 heterozygotes have not been fully described. In this article, we characterize the segregational aneuploidy generated by heterozygotes carrying eT1(III;V) and use one of the deletion homozygotes for telomere mapping.

MATERIALS AND METHODS

Strains and general methods: C. elegans var. Bristol strains were maintained as described previously (Brenner 1974). To observe large numbers of progeny over extended periods of time at a high power of magnification thinner media plates were used. These plates were identical to stock media plates in concentration and composition; however, the thickness was reduced from ∼7-2 mm to allow observation through the agar. Translocation nomenclature conforms to that of Rosenbluth and Baillie (1981). The translocation eT1(III;V) generates an Unc-36 mutant phenotype due to the breakpoint on chromosome III (Rosenbluth and Baillie 1981) and is genotypically always eT1[unc-36]. For brevity the unc-36 is not always written in the genotype. In addition to the N2 wild type, strains BC1265: + dpy-18 (e364) III; unc-46 (e177) V/eT1[unc-36] +; + and KR1054: dpy-5(e61) unc-13(e450) I; +/+ +; hT1[unc-42(e270)] V were used. Nematodes were maintained and all experiments performed at 20° unless otherwise stated.

Determination of aneuploid phenotypes: Hermaphrodites (20-30) were placed on a thin plate to lay eggs for 1-2 hr and then removed. Over the next 2 days, viable worms from this synchronized population were removed, leaving only the arrested aneuploid progeny. The progeny were examined for similarities and differences that could be used to group them into the six different phenotypic classes expected in the self-progeny of eT1 heterozygotes (Figures 1 and 2). Worms were observed using an IMT-2 inverted microscope (Olympus Corp., Lake Success, NY) with a differential interference contrast attachment and an Olympus BH2-HLSH lamp housing (12 V, 100 W), and Olympus TH3 power supply. This process was also aided by taking pictures (through the thin agar plate) of groups of freshly laid eggs over time and studying the pictures. Pictures of worms viewed with polarization and Nomarski optics were taken with an Olympus SC 35-mm camera and Ilford ISO 100/21° Delta black-and-white film. The embryos and larvae were grown on thin plates and, once arrested, were transferred in M9 buffer with a drawn-out pipette to a 5% agar pad (Sulston 1988). The coverslip was sealed to the slide using petroleum jelly.

Once phenotypes were established, brood counts were done to verify that the aneuploid phenotypes were in the expected numbers. Aneuploids were matched to genotypes either by observation of their arrest phenotypes or by specific crosses (see results). Aneuploid genotypes were verified using PCR.

Amplification of genomic DNA by PCR: A rapid way of detecting DNA deletions in homozygotes is the absence of a band under controlled conditions, using primers from the candidate region in a PCR reaction (Barsteadet al. 1991). In an adaptation of this method (F. Ho, personal communication), 10-20 BC1265 hermaphrodites (+ dpy-18; unc-46/eT1[unc-36] +; +) were transferred to thin plates to lay eggs for 1-2 hr, and then removed. The eggs were left overnight (14-20 hr), all viable progeny and larval arresting aneuploids were hatched, and only the embryonic arresting aneuploids remained. The hatched worms were removed from plates by washing with sterile water. The remaining embryos were treated with a small volume of Chitinase (Sigma, St. Louis; 20 mg/ml) for 1-2 min. A single embryo (or, in the case of spot eggs, four to five embryos) was transferred on fishing line from the plate to a 0.65-ml microcentrifuge tube containing 2.5 μl of worm lysis buffer (50 mm KCl, 10 mm Tris-HCl, pH 9, 1.0% Triton X-100, 1.5 mm MgCl2, 10 mg/ml Proteinase K). A drop of mineral oil was added to the tube and the contents were briefly spun in a microcentrifruge. The tubes were placed at -70° for at least 10 min to ensure breakage of the egg shell. The samples were incubated at 60° for 45 min to allow for protein digestion and then at 95° for 15 min to inactivate the Proteinase K. To amplify the genomic DNA, 50 pmol of forward primer 1, 50 pmol of reverse primer 1, 50 pmol of forward primer 2, 50 pmol of reverse primer 2, 2.5 μl 10× PCR buffer (Promega, Madison, WI), 1.25 mm MgCl2, 2.5 μl 10× dNTP mix (2 mm of each nucleotide), 2.5 units of Taq polymerase (Promega), and dH2O were added to each tube for a final volume of 25 μl. The mixture was amplified using a Perkin-Elmer Cetus (Norwalk, CT) DNA Thermal Cycler. The program for amplification was as follows: an initial 10 min denaturation at 94°, a 1-min annealing stage at 52°, and a 2 min extension stage at 72°, followed by 30 cycles of 1 min at 94°, 1 min at 52°, and 2 min at 72°. A final extension cycle followed with 1 min at 94°, 1 min at 52°, and 10 min at 72°. To distinguish the genotypes of the eT1 spot and eT1 no-spot embryos, two diagnostic primer pairs, unc-60-3/6 (McKim et al. 1994) and KRp64/65 (C. Thacker, unpublished results), were used. unc-60-3 (forward-TGACGA GAGCGAGATGAG) and unc-60-6 (reverse-CTTCTCCACAA CAAGCAT) amplify a 940-bp fragment from the unc-60 locus on the left end of chromosome V. KRp64 (forward-GGGAATG TCGAAAATCGCTG) and KRp65 (reverse-CCCTTGATTTA TAGCCACTC) amplify a 680-bp fragment from a putative gelsolin homologue, near the sup-5 locus on LGIII. The control primer pairs used for this analysis were KRp40/41 and KRp29/45; both pairs were designed for and are specific to the bli-4 locus on LGI (Thackeret al. 1995). KRp40 (forward-TGAAAT TCTTGGCCTCTAAC) and KRp41 (reverse-TGAAGCCACAGAAGTGAATC) amplify a 985-bp fragment and KRp29 (forward-TACTCACCCATATCAGTCAC) and KRp45 (reverse-ATACATATCCACCAACTGCT) amplify a 886-bp fragment. To distinguish between the hT1 spot and hT1 no-spot embryos, two diagnostic primer pairs, unc-60-3/6 (McKim et al. 1994) and KRp40/41, were used. The control primer pair used for this analysis was KRp64/65. The experiments were carried out in the same manner as for eT1. For the telomere experiment, primer pairs cTel 29.3/29.5 and cTel 3.5/3.3 were used (Wickyet al. 1996). cTel 29.3 (forward-CTAAGACCAATACCGCAAC) and cTel 29.5 (reverse-GTCTGAGACGTGATGT CTC) amplify a 270-bp fragment from the ribosomal gene cluster on LGI right. cTel 3.5 (forward-TCTAGACCCCCAGGATATG) and cTel 3.3 (reverse-GATCAAACAGAATGGGCTAT) were designed from the adjacent subtelomeric sequence of cTel 3 and amplify a 120-bp fragment. These two primer pairs were used in reactions with the following genomic templates: single N2 twofold embryos, single eT1-carrying no-spot embryos, 4-5 eT1-carrying spot embryos, and 4 hT1-carrying spot embryos, per reaction tube.

Figure 1.
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Figure 1.

—Photographs of the six classes of arrested progeny from an eT1 heterozygote (BC1265: + dpy-18 (e364) III; unc-46 (e177) V/eT1[unc-36] +; +). (A) An early arrested embryo that can be identified by the presence of a dark oval spot (nullo VL, tetra IIIL). (B) An early arrested no-spot embryo, which lacks a dark region (nullo IIIR, tetra VR). (C) An L1-arrested Dpy-like worm (haplo IIIR, triplo VL). (D) An L1 arrested worm that upon thin agar plates exhibited an Unc-36 shape (haplo IIIR, triplo VL). (E) An abnormal-appearing worm arrested near the time of hatching (haplo VL, triplo IIIR). (F) An L1-arrested worm, arrested near the time of hatching, but with a more elongated appearance than that shown in E (haplo VL, triplo IIIR). Bar, 10 μm.

RESULTS

The first step in characterizing aneuploids segregating from the eT1 heterozygote, + dpy-18; unc-46/eT1 [unc-36] +; +, was to examine the progeny and classify the phenotypes. The ratio of viable progeny expected is 1:4:1, Dpy-18 Unc-46 to Wild to Unc-36; dpy-18(III) and unc-46(V) show pseudolinkage because they are in the crossover-suppressed regions of this reciprocal translocation (Rosenbluth and Baillie 1981). In addition to the viable progeny, six classes of arrested aneuploids are expected. Initially, the arrested progeny were classified into phenotypic categories (Figure 1). A summary of our results is shown in Figure 2.

Identification of arresting embryos from eT1 heterozygotes: Two classes of arresting embryos could be distinguished. One class was recognized by the presence of a distinct dark spot measuring 10 × 15 μm. Under polarized light the dark spot was observed to coincide with the presence of gut granules, suggesting that the spot region is located in the developing gut region of the embryo (Figure 3). The second class of embryo lacked this dark feature, and thus the two classes were designated spot and no-spot.

Two of the segregational aneuploids are missing large portions of either LGIII or LGV. We predicted that deletions involving such a large portion of a chromosome would arrest as embryos. Thus, we anticipated that one of the spot and no-spot arresting embryonic classes would be a deletion of LGIII to the right of the eT1 breakpoint, and that the other class would be a deletion of LGV to the left of the eT1 breakpoint. To gain more information about which chromosomal region was missing in each class of embryo (either LGIII right or LGV left), we examined the progeny from heterozygotes of a second translocation involving LGV left. hT1 is a reciprocal translocation involving LGI and LGV, in which crossing over is suppressed from the breakpoints leftward to the end of the chromosomes, on LGI and LGV (McKim et al. 1988). In the progeny of hT1 heterozygotes, arrested spot embryos were also observed. It seemed most likely that this arrested spot embryo was a deletion of LGV left, because this region was involved in both the hT1 and the eT1 rearrangements.

Confirmation of aneuploid embryos from eT1 heterozygotes: PCR analysis was used to test for the presence of DNA in specific regions of the arrested spot and no-spot embryos. Embryos were picked individually into reaction tubes on the basis of a visible phenotype, ensuring that either a spot or no-spot embryo was used for template DNA. Genomic DNA from spot embryos was used in PCR reactions containing a primer pair unique to LGIII right, KRp64/65, and another primer pair unique to LGV left, unc-60-3/6. A single 680-bp fragment was produced, corresponding in size to the product expected from the primer pair unique to LGIII right (Figure 4, lane C). When N2 DNA was used as template, both fragments were observed (Figure 4, lane B). As an additional control, the LGIII right primer pair was used with a primer pair from LGI, Krp40/41. With spot embryo DNA as template, two fragments, 680 bp and 995 bp, were produced, corresponding in size to those expected for the LGIII and LGI primer pairs, respectively (data not shown). Thus, we concluded that LGV left DNA is missing in the arrested spot embryo.

Figure 2.
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Figure 2.

—Punnet square summarizing the progeny of eT1 heterozygotes. Chromosome III is drawn in black; chromosome V in red. The recombining end of each chromosome (homologue recognition region) is marked with an asterisk. The normal chromosomes carry dpy-18 (e364) on III, and unc-46 (e177) on V (marked with a vertical line). The eT1 chromosomes have the wild-type alleles of these genes. eT1 breaks in unc-36.

Figure 3.
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Figure 3.

—Arrested embryos from an eT1 heterozygote (BC1265: + dpy-18 (e364) III; unc-46 (e177) V/eT1[unc-36] +; +). (A) Wild-type embryo, (B) spot embryo, (C) no-spot embryo. Bar, 10 μm.

Genomic DNA from the no-spot embryo was also tested. Using the LGIII right primer pair along with the LGV left primer pair, a single fragment of 940 bp was observed (Figure 4, lane D). This was the expected size for the LGV left primers. In a control reaction, using the LGV left primer pair with a primer pair from LGI, KRp45/29, two products were seen, 940 bp and 880 bp, respectively. As a further control, combinations of pairs of primers were tested using DNA from a wild-type (N2) embryo as the template. In these experiments, two bands for each reaction were observed. The no-spot embryo contains template for the LGV primers, but not for LGIII. Thus, we concluded that the spot embryo is a homozygous deletion of LGV left (of the eT1 breakpoint) and that the no-spot embryo is a homozygous deletion of LGIII right (of the eT1 breakpoint).

Figure 4.
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Figure 4.

—PCR results for embryos from eT1 heterozygotes. Template DNA prepared from N2 wild-type embryos (B); spot embryos (C); and no-spot embryos (D). A and E contain 100-bp ladder markers. The top fragment corresponds to the expected size (940 bp) for primers unc-60-3/6 from LGV. The lower band corresponds to the expected size (680 bp) for KRp64/KRp65, near sup-5 on LGIII. Arrows show expected fragment sizes.

Characterization of the remaining aneuploids: Four additional classes of aneuploids are expected in the progeny of eT1 heterozygotes, those that were homozygous for either dpy-18, unc-46, or eT1[unc-36], and those that had no homozygous marker (for simplicity referred to as “wild”). We observed one class of arrested worms with a pointed or cone-shaped head, often found lying in a kinked position. On the thin plates (see materials and methods), the movement of these worms could be observed, and was very slow. Backward motion appeared to be smoother than forward motion, as though the head region were paralyzed. These worms resembled homozygous eT1 homozygotes, which have an Unc-36 phenotype. In addition there were arrested progeny that looked somewhat dumpy. These worms had a wide head region and resembled Dpy-18 (Figure 1C). A third category did not have any noticeable dumpy or uncoordinated phenotype. They were tested for response to a nose-tap. Unc-46 shrinks backward in response to a nosetap. However, this test was inconclusive as many progeny did not move or made ambiguous movements in response to tapping. A fourth category were worms that looked as if they had died soon after hatching. These looked like prematurely hatched worms that had begun to disintegrate (Figure 1E). Often, they did not hatch and were observed as threefold embryos. Either of the latter two groups could be the arrested wild or Unc-46 phenotypic category.

The arrested wild and Unc-46 phenotypes could not be assigned by observation. Crosses to determine their genotypes were done. In the first cross, dpy-18; unc-46 homozygotes were crossed to BC1265 males (+ dpy-18; unc-46/eT1[unc-36] +; +). In the progeny of this cross, arrested Dpy-18 and Unc-46 are expected, but not arrested Wild or Unc-36. Arrested Dpy-18 and a second aneuploid class were observed (Figure 5, A and B). In the second cross, homozygous eT1[unc-36] hermaphrodites were mated with BC1265 males. In the progeny of this cross, arrested eT1[Unc-36] and Wilds were expected. A class of worms described as having “hatched and died” were observed, as well as the predicted arrested Unc-36 (Figure 5, C and D). Thus, the hatched and died group corresponded to the arrested wild group. The group with no distinctive phenotype other than developmental arrest was the Unc-46 category (Figure 5B). In comparison with the hatched and died phenotype, these Unc-46 marked worms were somewhat more elongated.

Figure 5.
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Figure 5.

—Results of crosses confirming genotypes of the eT1 aneuploid worms. Progeny resulted from crossing dpy-18; unc-46/dpy-18; unc-46 hermaphrodites to heterozygous eT1 males. The progeny would have only the Dpy-18 and Unc-46 aneuploids. (A) Arrested Dpy-like worms; (B) a non-Dpy class which must be the Unc-46 aneuploids. Progeny resulted from a cross between eT1 homozygous hermaphrodites and heterozygous eT1 males. The progeny from this cross would have only the Unc-36 and wild (nonmarked) aneuploids. We observed the worms referred to as “hatched and died” (C) and worms that look Unc-36 (D), making the hatched and died category the unmarked class of aneuploids. Bar, 10 μm.

Identification of aneuploid embryos from hT1 heterozygotes: Arrested embryos in the progeny of hT1 heterozygotes were examined as described for those from eT1 heterozygotes. However, the hT1 homozygote is inviable, making three classes of arresting embryos. Two of the embryos arrested in early gastrulation, before the comma stage and the third embryonic class arrested at the three-fold stage. One class of the earlier arresting embryos had the spot phenotype, which we expected to be missing LGV left. We expected the other early arresting embryo would be missing LGI left. PCR was performed to test these predictions (data not shown). Genomic DNA from the arrested spot embryo was used in a PCR reaction with two primer pairs, one unique to LGIII right, KRp64/65, and another unique to LGV left, unc 60-3/6. A single 680-bp fragment was produced, corresponding to the size expected for the LGIII right primer pair. As a control, spot embryo DNA was tested using the LGIII right primer pair with a primer pair from LGI, KRp40/41. In this reaction, two fragments, 680 and 995 bp, were produced, the expected sizes for the LGIII and LGI primer pairs, respectively. The tested spot embryos were missing LGV DNA. Similarly, the no-spot embryo was tested. In reactions using the LGV left primer pair along with the LGIII right control primers, two fragments were produced. The length of the fragments corresponded to the expected 940-bp and 680-bp products for the LGV and LGIII primers. When the control primer pair from LGIII was used in the same reaction as a primer pair from LGI left (KRp40/41), only one fragment was produced. The resulting 680-bp fragment corresponded to the expected product for the LGIII control primer. In control experiments using N2 embryonic DNA, two products of the expected sizes were seen. Thus, the arrested spot embryo from hT1 heterozygotes has a similar genotype as the arrested spot embryo from eT1 heterozygotes and is deleted for a large portion of the left end of LGV. A second early arresting embryo, lacking a spot phenotype, was deleted for the left portion of LGI. The later arresting embryo is assumed to be the hT1 homozygote.

Mapping a telomeric clone using the tip deletions: Using DNA from the deletion homozygotes, we tested primer sets corresponding to the cloned telomere probes. DNA from eT1 spot embryos, eT1 no-spot embryos, hT1 spot embryos, and N2 embryos was used in PCR reactions. Control primers from the ribosomal cluster on LGI produced a 270-bp band in all reactions. cTel3 primers (cTel 3.3/3.5) produced a 120-bp fragment in N2 and eT1 no-spot embryo DNA, but no PCR product was produced using the cTel3 primers from arrested spot embryo DNA (Figure 6, lanes A-C). We repeated this experiment using spot and no-spot embryos from hT1 heterozygotes, and observed that the spot embryos from this translocation also lacked the cTel3 sequences (Figure 7). We conclude that the clone cTel3 is derived from the left (unc-60) end of chromosome V.

DISCUSSION

Rosenbluth and Baillie (1981) described a homozygous viable reciprocal translocation, eT1, which consists of two aberrant chromosomes. In heterozygotes, the chromosome referred to as eT1(III) recombines with and disjoins from the normal LGIII, and the other chromosome, eT1(V), recombines with and disjoins from the normal LGV. No adjacent-2 disjunction is observed. Extrapolating from the work of Hawley (1980), Rosenbluth and Baillie (1981) suggested that the segregational patterns were determined by pairing in the recombinationally active portions. Furthermore Rosenbluth and Baillie (1981) showed that the regions that crossed over disjoined from each other. The pattern of segregation was predicted to result in the observed viable offspring plus a majority (10/16) of aneuploid progeny that had inherited a nondiploid chromosome complement. Six classes of aneuploid genotypes were predicted, including homozygous deletions from the site of the eT1 breakpoint to the end of the chromosome. Using PCR analysis, we have demonstrated the existence of the predicted LGIII right and LGV left deletions, which arrested as embryos.

Figure 6.
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Figure 6.

—PCR using cTel primers. cTel 29.3/29.5 amplified a 270-bp fragment from the ribosomal cluster on LGI right (upper band); and cTel 3.5/3.3 amplified a 120-bp fragment (lower band). Template DNA was prepared from embryos that were either spot (A, B, C), no-spot (D, E, F), or wild-type N2 (G, H).

We were able to distinguish reproducibly two classes of arrested embryos by the presence of a dark spot. The presence of the dark spot correlated with deletion of chromosome V left (nullo VL, with four copies of IIIR). The other, no-spot, embryo was nullo IIIR, with four copies of VL. The next two most severe phenotypes were both haplo VL and triplo IIIR, and arrested near the time of hatching. One of these aneuploids contained one normal chromosome III, one eT1(III), and two copies of eT1(V). The other contained two normal chromosome IIIs, one normal chromosome V, and one eT1(V). This second aneuploid was haplo for unc-46(e177), containing no wild-type allele and one copy of the mutant allele. Phenotypically this aneuploid was somewhat more elongated than the nonmarked aneuploid. The other two categories of aneuploids were haplo IIIR-triplo VL. One lacks a wild-type allele of dpy-18, while the other lacks a wild-type allele of unc-36. These phenotypes are expressed even though none of the aneuploids developed beyond the first larval stage.

Figure 7.
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Figure 7.

—PCR results for embryos from hT1 heterozygotes using cTel primers. Primers cTel 29.3/29.5 and cTel 3.5/3.3 amplified only the 270-bp fragment (expected for the LGI primer pair) using template DNA from spot embryos. Both the 270- and the 120-bp fragments were observed using template DNA from no-spot embryos from hT1 heterozygotes.

The segregational aneuploids studied here result from the meiotic behavior of translocations. McKim et al. (1988) extended the hypothesis of Rosenbluth and Baillie (1981) regarding the pairing, crossing over, and disjunction patterns of eT1 heterozygotes to other reciprocal translocations. These authors proposed that one end of each chromosome, designated the homolog recognition region, determined pairing partners and subsequent meiotic events. In this study, we examined two reciprocal translocations. In the progeny of heterozygotes for hT1, a translocation involving chromosomes I and V, three classes of arrested embryos were observed. One of these had a recognizable dark spot. In the case of hT1, as for eT1, the dark spot correlated with deletion of chromosome V left. The common feature of hT1 and eT1 is the involvement of chromosome V left, and thus the dark spot most likely results from the absence of gene(s) in LGV left and not from an overexpression of genes in LGIII right or LGI left.

The dark spot coincides with the localization of birefringent gut granules observed in wild-type embryos. The birefringence in the spot embryos is much more intense than that of wild-type embryos, suggesting an accumulation of refractile tryptophan catabolites possibly correlating with cell lysis. A persistent cell death phenotype was observed by Ahnn and Fire (1994) for some deficiencies of this region and may correlate with our spot phenotype. Ahnn and Fire (1994) and Storfer-Glazer and Wood (1994) studied the arrest stages for several deletions of LGV and reported the earliest to be before the comma stage. The distance from the eT1 (or hT1) breakpoint to the end of the chromosome is larger than any of the previously studied deletions. We would expect the arrest stage for the spot embryo to be as early or earlier than any of the smaller deletions, depending upon the cause of the embryonic arrest. In our study, the spot embryos arrested in the second stage of embryogenesis, gastrulation, as described in Wood (1988). Gastrulation begins approximately 100 min after first cleavage and continues for approximately 250 min. The E cell lineage begins differentiating as the gut at 60 min after first cleavage. In the arrested spot embryo, the gut cells appear to be clumped together. In the no-spot embryo, however, the gut cells appear to be dispersed and we estimated that the no-spot embryo had developed further than the spot embryo. Arrested no-spot embryos from hT1 heterozygotes also appeared to develop further than the arrested spot embryos.

Wicky et al. (1996) have cloned 11 of the 12 C. elegans telomeres and for each clone designed primer sets that amplify small fragments of unique subtelomeric sequence. Two telomeric clones were mapped in the analysis of Wicky et al. (1996), the right end of chromosome I and the left end of the X chromosome. In this article, we have mapped a third clone, cTel3, to LGV left. In this study, it was not feasible to test the embryos with all the telomere probes, to identify the clones corresponding to chromosome IIIR or chromosome IL. Theoretically, translocations could be used to position those telomere clones that map to the crossover-suppressed ends of chromosomes involved in reciprocal translocations.

The deletion embryos used in this analysis arise by meiotic segregation and differ fundamentally from deletions induced by mutagenesis, which contain telomeric sequences at their ends. Induced deletions are “capped” either by rejoining to their native telomere or by de novo telomere formation (Wickyet al. 1996). In contrast to induced deletions that appear to be at the end of the genetic map, but for which the composition of the DNA capping the deletion is not always known, these deletion homozygotes lack the entire end of the chromosome because it has segregated away. Thus, the DNA present in these embryos could be compared to that present in induced deletions to help characterize the composition of other terminal deletions. Deletion homozygotes from translocation heterozygotes provide an additional source of tip deletions, which can be characterized using PCR analysis of single embryos. The mapping of the telomeric clone described in this analysis is one example of the use of aneuploids from translocation heterozygotes as a source of terminal deletions to increase our understanding of the structure and function of chromosome ends in C. elegans.

Acknowledgments

The authors thank Mark Edgley, Raja Rosenbluth, Bob Johnsen, and David Baillie for stimulating discussions and comments on the manuscript. This research was supported by the National Center for Research Resources of The National Institutes of Health (United States) and the Natural Sciences and Engineering Research Council (Canada).

Footnotes

  • Communicating editor: R. S. Hawley

  • Received March 27, 1998.
  • Accepted July 13, 1998.
  • Copyright © 1998 by the Genetics Society of America

LITERATURE CITED

  1. ↵
    1. Ahnn J.,
    2. Fire A.
    , 1994 A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans. Genetics 137: 483–498.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Barstead R. J.,
    2. Kleiman L.,
    3. Waterson R. H.
    , 1991 Cloning, sequencing, and mapping of an alpha-actinin gene from the nematode Caenorhabditis elegans. Cell Motil. Cytoskeleton 20: 69–78.
    OpenUrlCrossRefPubMedWeb of Science
  3. ↵
    1. Brenner S.
    , 1974 The genetics of Caenorhabditis elegans. Genetics 77: 71–94.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Epstein H. F.,
    2. Shakes D. C.
    1. Edgley M.,
    2. Baillie D. L.,
    3. Riddle D. L.,
    4. Rose A. M.
    , 1995 Genetic balancers, pp. 148–184 in Caenorhabditis elegans: Modern Biological Analysis of an Organism, edited by Epstein H. F., Shakes D. C.. Academic Press, San Diego.
  5. ↵
    1. Hawley R. S.
    , 1980 Chromosomal sites necessary for normal levels of meiotic recombination in Drosophila melanogaster. I. Evidence for and mapping of the sites. Genetics 94: 625–646.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Herman R. K.
    , 1978 Crossover suppressors and balanced recessive lethals in Caenorhabditis elegans. Genetics 88: 49–65.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Johnsen R. C.,
    2. Baillie D. L.
    , 1988 Formaldehyde mutagenesis of the eT1 balanced region in Caenorhabditis elegans. Mutat. Res. 201: 137–147.
    OpenUrlPubMed
  8. ↵
    1. Johnsen R. C.,
    2. Baillie D. L.
    , 1991 Genetic analysis of a major segment [LGV(left)] of the genome of Caenorhabditis elegans. Genetics 129: 735–752.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. McKim K. S.,
    2. Howell A. M.,
    3. Rose A. M.
    , 1988 The effect of translocations on recombination frequency in Caenorhabditis elegans. Genetics 120: 987–1001.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. McKim K. S.,
    2. Matheson C.,
    3. Marra M. A.,
    4. Wakarchuk M. F.,
    5. Baillie D. L.
    , 1994 The Caenorhabditis elegans unc-60 gene encodes proteins homologous to a family of actin-binding proteins. Mol. Gen. Genet. 242: 346–357.
    OpenUrlCrossRefPubMedWeb of Science
  11. ↵
    1. Rosenbluth R. E.,
    2. Baillie D. L.
    , 1981 Analysis of a reciprocal translocation, eT1(III;V) in Caenorhabditis elegans. Genetics 99: 415–428.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Rosenbluth R. E.,
    2. Cuddeford C.,
    3. Baillie D. L.
    , 1983 Mutagenesis in Caenorhabditis elegans I: a rapid eukaryotic mutagen test system using the reciprocal translocation eT1(III;V). Mutat. Res. 110: 39–48.
    OpenUrl
  13. ↵
    1. Rosenbluth R. E.,
    2. Cuddeford C.,
    3. Baillie D. L.
    , 1985 Mutagenesis in Caenorhabditis elegans II: a spectrum of mutational events induced with 1500 R of gamma-radiation. Genetics 109: 493–511.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Rosenbluth R. E.,
    2. Rogalski T. M.,
    3. Johnsen R. C.,
    4. Addison M.,
    5. Baillie D. L.
    , 1988 Genomic organization in Caenorhabditis elegans: deficiency mapping on linkage group V(left). Genet. Res. 52: 105–118.
    OpenUrlWeb of Science
  15. ↵
    1. Storfer-Glazer F. A.,
    2. Wood W. B.
    , 1994 Effects of chromosomal deficiencies on early cleavage patterning and terminal phenotype in Caenorhabditis elegans embryos. Genetics 137: 499–508.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Wood W. B.
    1. Sulston J. E.
    , 1988 Cell lineage, pp. 123–155 in The Nematode Caenorhabditis elegans, edited by Wood W. B.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  17. ↵
    1. Thacker C.,
    2. Peters K.,
    3. Srayko M.,
    4. Rose A. M.
    , 1995 The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases, essential for early development and adult morphology. Genes Dev. 9: 956–971.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Wicky C.,
    2. Villeneuve A. M.,
    3. Lauper N.,
    4. Codourey L.,
    5. Tobler H.,
    6. et al.
    , 1996 Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 93: 8983–8988.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Wood W. B.
    (Editor), 1988 The Nematode Caenorhabditis elegans, Vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Volume 150 Issue 3, November 1998

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Mapping a Telomere Using the Translocation eT1(III;V) in Caenorhabditis elegans

K. A. Adames, Jocelyn Gawne, Chantal Wicky, Fritz Müller and Ann M. Rose
Genetics November 1, 1998 vol. 150 no. 3 1059-1066
K. A. Adames
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Mapping a Telomere Using the Translocation eT1(III;V) in Caenorhabditis elegans

K. A. Adames, Jocelyn Gawne, Chantal Wicky, Fritz Müller and Ann M. Rose
Genetics November 1, 1998 vol. 150 no. 3 1059-1066
K. A. Adames
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Jocelyn Gawne
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