Hoskins R (1987) Worm Breeder's Gazette "unc-30 on the map."

Hoskins R

[Worm Breeder's Gazette, 1987]

The unc-30 gene has been found to lie on the physical map, in a contig previously shown by J. Yuan to span the ced-3 locus(CSH 1987). unc-30 recombinants were picked from the progeny of unc-31(e928)unc- 30(e191)/BO parents and used to map BO/N2 DNA polymorphisms found in the ced-3 contig. The data indicate that unc-30 is surprisingly close to ced-3.Two polymorphisms have been mapped. They have been designated eP56 and eP57, and are recognized by hybridization with the cosmids ZK987 and C43A2, respectively. They map as follows: unc- 31[8]eP56[4]ePS[1]unc-30. A right-hand limit on the position of unc- 30 has been established by Yuan. She has mapped a BO-specific Tc1 to the right of ced-3 in the cosmid MMMC1. The physical distance between unc-30 and ced-3 must therefore be less than 120kb of DNA; the genetic distance between them is 0.3-0.5%. It should now be possible to clone the unc-30 gene. Attempts toward this end have been hampered by the potpourri of repeat sequences in cosmids to the right of eP57. Several DNA polymorphisms have been found in EMS, [32P], and spontaneous BO unc-30 alleles.

Beckenbach KA et al. (1988) Worm Breeder's Gazette "strain identification using mitochrondria RFLD's."

Rose AM, Beckenbach KA, Baillie DL

[Worm Breeder's Gazette, 1988]

We have investigated the possibility of using RFLD's (Restriction Fragment Length Differences) of a mtDNA fragment to distinguish the various C. elegans strains. Ann Rose's lab cloned the 5kb EcoRI restriction fragment (pCeh109) of the mitochondria DNA. Total DNA from N2, BO, PA-2, GA-1, and N2(DR) was isolated, restricted, fractionated on an agarose gel, and transferred to nitrocellulose. The filter was then probed using [32p] labelled pCeh109. The restriction enzymes we used were AccI, AluI, AvaII, DdeI, DraI, HaeIII, HincII, HinfII, and RsaI. Six of the nine enzymes used showed an RFLD that easily distinguished Pa2 from the others, and two showed a possible difference between N2 and BO. Since PA-2 shows an RLFD with so many of the restriction enzymes, it is possible that the mtDNA of this strain has some sort of rearrangement. The nature of these RLFD's and the ones between N2 and BO are currently being investigated.

Beckenbach KA et al. (1984) Worm Breeder's Gazette "the cloning of the region around unc-22."

Beckenbach KA, Rose AM, Baillie DL

[Worm Breeder's Gazette, 1984]

The region around unc-22, flanked by unc-43 on the left and unc-31 on the right, has been intensively studied in our laboratory over the last eight years using conventional genetic analysis. We have now embarked upon a molecular analysis of this same region. We have at present identified and recovered about 300 kb of DNA that lies within this two map unit interval. Tc1 induced restriction fragment length differences closely linked to unc-22 were identified in the N2 and BO strains. DNA was prepared from a strain constructed by serial backcrossing of N2/BO heterozygotes to N2 males (where the BO chromosome IV was marked with a EMS induced unc-22 allele). After each cross the unc-22 marked BO chromosome was recovered homozygous. This procedure was repeated six times. A lambda gt10 EcoRI library was constructed and subsequently screened for Tc1-containing sequences. These phage were isolated and their genomic fragments subcloned into pUC13 or pUC19 plasmid vectors. The flanking sequences were recovered by EcoRV digestion followed by blunt end ligation and recloning of the Tc1 deleted plasmid. These plasmids provided convenient probes for the mapping of the associated N2/BO RFLD. Three factor mapping was carried out using the marker pairs unc-43 unc-31 to map the plasmid-defined RFLD. The resulting newly-defined molecular sites are shown on the accompanying figure. The plasmids which defined each site were then used to isolate genomic clones from our lambda Charon 4 library (prepared by T. Snutch). About 25-30 kb of flanking sequence was recovered for each probe. These lambda phage were in turn checked for overlap with existing cosmids in John Sulston's genomic mapping collection. [See Figure 1]

Donati LM et al. (1984) Worm Breeder's Gazette "a hybrid dysgenesis-like phenomenon in C elegans."

Baillie DL, Donati LM

[Worm Breeder's Gazette, 1984]

We are studying the unc-22 f linkage group IV . Our experiments make use of R. Horvitz's balancer nT1. In one experiment devised to study the effect of heat shock mutagenesis in C. elegans , we noticed that when Bergerac (BO) hermaphrodites were mated to Bristol (N2) males, a high rate of mutation was detected both in our control and in our experimental. unc-22 BO hermaphrodites (EMS induced by Teresa Rogalski) were mated to unc-22 1 N2 males. Single wild-type F1's were set and their progeny analyzed for the absence of Unc-22 individuals. Of 537 chromosomes screened, 24 lethal bearing chromosomes were recovered (4-5%). This is an increase of approximately 50 fold over the spontaneous rate of lethal induction in the Bristol strain (Rosenbluth et. al., Mutation Research 110:39-48). Several of these mutations have been mapped relative to unc-22 . One maps 8.5 map units from unc-22 , another maps 4 map units away. Of interest are three independently derived mutations which fail to complement one another and let-52 and appear tightly linked to unc-22 . These mutations may either be point mutations of let-52 or may be chromosomal rearrangements in this region. We are able to detect lethal bearing chromosomes in the progeny of the F1 suggesting that the mutations are fixed in the F1. Therefore, two possibilities exist as to the nature of these mutations. The unc-22 BO strain may have an unusually high spontaneous mutation rate. Alternatively, the high mutation rate may be due to a hybrid dysgenesis-like phenomenon, occurring sometime after fertilization and prior to the development of the germline of the F1. If the unc-22 BO strain had an endogenous high spontaneous mutation rate, one would expect a large number of clustered mutational events. We have evidence of one such event. We are in the process of establishing a N2/BO let-52 strain which has been repetitively backcrossed to Bergerac. Examination of this strain may allow us to detect reversion. Also of interest is the distribution of these lethals. EMS induced lethals seem to be primarily to the left of unc-22 . There is only one EMS induced allele of let-52 , while there are three N2/BO interstrain cross induced alleles. The molecular basis of these mutations is being investigated. If these mutations are due to transposable element insertions, either Tc1 or some other element, it will be possible to isolate insertional mutations in genes of interest. These molecularly marked strains will be invaluable for the cloning and molecular identification of specific genes.

Rose AM et al. (1983) Worm Breeder's Gazette "RFD's as probes for cloning."

Beckenbach KA, Rose AM, Mawji NR, Baillie DL, Nelson DW

[Worm Breeder's Gazette, 1983]

We have been screening for restriction fragment differences (RFDs) between the N2 and Bo strains as described by Emmons, et al., 1979. Random N2 fragments with EcoRI and HindIII ends were cloned into pBR322, isolated, and purified. Using these fragments as P nick translated probes, RFDs were detected about one per six tested probes. We are in the process now of accumulating and mapping a set of these probes. It is our hope to establish a reasonably large collection of mapped RFD probes that might be utilized in the cloning of genes with no biochemically identified products. Towards this end we have mapped four such RFDs: One of these pGe s18 has been further mapped to the left of unc-13 ( I). Terry Snutch has screened our Charon 4 library for corresponding lambda clones of this region, which we have restriction mapped and subcloned for chromosome walking. We hope that characterization of the DNA adjacent to pCe s18 will allow us to isolate some of the genes in the unc-15 region described by Rose and Baillie, 1980. Interestingly, probe pCe s18 when hybridized against N2, Bo, and FF shows the N2 banding pattern. We would welcome information on the relationship (if any) between Bo and FF. [See Figure 1]

Nelson DW et al. (1984) Worm Breeder's Gazette "mapping a 5S DNA cluster."

Honda BM, Nelson DW

[Worm Breeder's Gazette, 1984]

The recombinant phage RW70-4 (obtained from Bob Waterston) carries 4- 5 kb of Bristol (N2) genomic sequence in addition to the 1 kb 5S DNA repeat. We previously reported that the 2.2 kb BamHI fragment immediately flanking this 5S DNA cluster is shifted to a pair of BamHI fragments (2.5 and 2.6 kb) in the Bergerac (BO) genome. This RFLD was shown to be loosely linked to dpy-11(V).We have left-right positioned this RFLD with respect to several linkage group V markers as shown below. The results to date confirm the linkage group V assignment and place this RFLD to the right of unc-76. This correlates well with the in situ hybridization results placing the 5S RNA gene cluster towards the end of the chromosome (D. Albertson EMBO J. 3 p1227-1234). [See Figure 1] While constructing the recombinant strains to complete these mapping experiments (still in progress), we noticed a reduction in the recombination distance separating dpy-21 and unc-51 in the N2/BO heterozygote as compared to the N2/N2 heterozygote (p=0.06 and 0.09 respectively). All other distances were essentially identical (ie: unc-76 , dpy-21). The sample size is not yet sufficient to lend statistical significance to this reduction and we are repeating the experiment anticipating that this may reflect a small rearrangement in this region of the BO genome relative to the N2 standard. Such a rearrangement may be related to the RFLD we have identified.

Mori I et al. (1984) Worm Breeder's Gazette "a needle in a haystack?"

Waterston RH, Moerman DG, Mori I

[Worm Breeder's Gazette, 1984]

Our studies on unc-22 and those of D. Eide and P. Anderson on unc- 54 suggest that high Tc1 copy number per se is not sufficient to induce transposition. However, we also know from our genetic studies on unstable unc-22 mutations that transposition into unc-22 is probably controlled by more than one region present in the BO strain ( Moerman and Waterston, Genetics in press). In light of these two observations we are attempting to isolate a transposition 'factor'. Here we use factor as it has been described for P in D. melanogaster. To monitor the activity of such a factor we have used the reversion frequency of an unstable unc-22 mutation, st136. In the BO strain, RW7002, st136 reverts at a frequency of 2 x 10+E-3, but in RW7012, a primarily Bristol derivative of RW7002, st136 reverts at a frequency of less than 10+E-7. We have constructed 12 isogenic strains of mixed Bristol-Bergerac genomes from hybrids of RW7012 and BO, and these strains exhibit a range of reversion frequencies from about 10+E-3 to probably less than 10+E-6. The observation of a rather large 'low reversion frequency' class, about 25% of the strains, suggested there may actually be very few factors. An attractive hypothesis is that the factors may be a subset of the Tc1 elements present in BO. We examined these new st136 Bristol- Bergerac strains for Tc1 copy number, and found them to differ by as much as 3-fold. No correlation between a strains Tc1 copy number and its reversion frequency was observed. One strain, RW7037, has a relatively low Tc1 copy number (tentatively less than 100 copies) and also a high reversion frequency. We think it may be possible to use derivatives of RW7037 to map genetically a single factor. If we are lucky this factor will be homologous to Tc1.

Egilmez NG et al. (1993) Worm Breeder's Gazette "Quantitation of Tc1 Elements in Various Strains of C. elegans"

Egilmez NG, Shmookler Reis RJ

[Worm Breeder's Gazette, 1993]

We have employed the transposable element Tc1 as a polymorphic marker in order to define the evolutionary relationships between strains of C elegans. We first determined the number of Tc1 elements in strains N2 ,PA2 ,C12 aand Ga1 ,reported to be low-copy number strains and RC301 ,TR403 ,DH424 ,RW7000 and BO, described as high-copy variants (1). N2 is commonly referred to as having 30 Tc1s in its genome, while the values reported for BO vary between 300 and 500. No specific values have been reported for the other strains mentioned above. We utilized two different approaches to determine the Tc1 copy numbers. Initially, genomic DNAs from each strain were digested with EcoRI and separated on large, slow-running 0.8% agarose gels (23cm, 20V, 96 hours). Southern blots of these gels were then probed with a 1.6 kb cloned Tc1 element (2). Tc1 elements of the low-copy number strains could be resolved sufficiently well on these blots to allow direct enumeration from the autoradiographs. The results obtained from these blots for the low-copy strains are shown in Table 1. Tc1 copy numbers varied between 26 and 30 and the overall patterns were similar with minor differences between strains. RC301 ,contrary to previous reports (1), appeared to be a low-copy strain, containing 26 Tc1 bands. Re-analysis of fresh RC301 stocks, re-ordered from the Caenorhabditis Genetics Center and kindly provided by J. Hodgkin (MRC, Cambridge, England), confirmed our initial results. The band patterns of GA1 and N2 were identical. Other low-copy strains were distinct from N2 and from each other, in their Tc1 band patterns, but retained 21-22 bands with mobilities indistinguishable from N2 bands. C12 aand PA2 were clearly different from N2 but were nearly identical to each other differing at only one band. RC301 appeared to be the most distant relative of N2 based on the number and pattern of its Tc1 elements. DNA dot-blots were utilized to analyze the Tc1 copy numbers of the high-copy strains. Genomic DNAs from N2 ,RC301 ,TR403 ,DH424 ,RW7000 and BO were blotted at four different dilutions and were probed with the cloned Tc1 element. The values for DNA loads were corrected by probing the same blot with a cloned single-copy gene fragment (2). The blots were analyzed by direct phosphor-imaging of -emissions on a Phosphor Imager Computing Densitometer (Molecular Dynamics). Intensities of phosphor-images were integrated directly using ImageQuant software and the results are summarized in Figure 1. All values were within the linear range of the phosphor- screen extending over a 105 range of signal intensities thus ensuring reliable quantitation. Using RC301 as a reference standard, at 26 Tc1s ,the Tc1 copy numbers for the strains TR403 ,DH424 ,RW7000 and BO were estimated to be 205, 260, 410 and 494 respectively, based on least-squares linear regression. The slope for RW7000 ,a separately maintained stock of Bergerac-BO, has a large standard error and is not significantly different from that of BO. Nevertheless, the difference could be genuine, since different levels of Tc1 -transpositionmay have accumulated in these two stocks of an active mutator strain. In all other cases, the lines fit the data well and thus the values determined should be highly accurate. References: (1) Cassada, R. WBG 9(3):29, 1986; Hodgkin, J. WBG 10(2):140-141, 1988; Hodgkin, J. WBG 11(5):60, 1991. (2) Egilmez, N.K. & R.J. Shmookler Reis, manuscript in preparation.

Radice AD et al. (1988) Worm Breeder's Gazette "analysis of Tc1 transcription."

Sherif NE, Emmons SW, Radice AD

[Worm Breeder's Gazette, 1988]

We have isolated polyadenylated RNA from a series of mutator and non- mutator strains with various rates of Tc1 germline transposition, and analyzed these in Northern hybridizations using a Tc1-specific RNA probe. RNA from the following strains has been analyzed: Bristol (30 copies of Tc1/inactive), RW7414 (80 copies/inactive), RW7406 (80 copies/active), Bergerac(BO) (300 copies/active), DH424 (300 copies/inactive), and TR679 (>300 copies?/very active). The two RW strains were constructed in the Waterston laboratory by a series of backcrosses of Bergerac to Bristol. One, RW7406, retains the mut-4 locus on LGI, whereas the other, RW7414, has no mutator. An actin III- specific probe was used to normalize the amounts of polyadenylated RNA present in each lane. We detect a Tc1 transcript of 1.3 kb in all of the above strains except Bristol. There is also much additional hybridization in most of the lanes, including a possible transcript of 3.8 kb in TR679. However, there is also significant cross hybridization of the Tc1 probe (but not the actin probe) to ribosomal RNA, even in poly A+ RNA preparations, and even after hybridization and washing under stringent conditions. This complicates the interpretation of the additional bands. The amount of the 1.3 kb Tc1 transcript appears to be greater in strains where Tc1 is active in the germline. The relative levels, normalized to the amount in Bergerac(BO), are as follows: Bristol, <0. 1 (undetectable); DH424, 0.25; RW7414, 0.5; RW7406, 1.5; Bergerac (BO), 1.0; TR679, 5. The level of hybridization in TR679 is high, about half the level of hybridization of our actin probe to the three major actin mRNA's. Dividing by the number of Tc1 elements in each strain, and once again normalizing to the level in Bergerac(BO), the levels of Tc1 transcript per Tc1 element are: Bristol (inactive), <1; DH424 ( inactive), .25; RW7414 (inactive), 1.89; RW7406 (active), 5.7; Bergerac(BO) (active), 1.0; TR679 (active), 5.0. These data are preliminary and subject to considerable error, including errors in the estimates of the number of Tc1 elements in the various strain backgrounds, but they suggest that activation of Tc1 transcription may accompany activation of Tc1 transposition in the germline. In the Tc1 sequence of Rosenzweig et al., the distance between the putative TATA and polyadenylation sequences of Tc1 is 1090 bp. We are presently mapping the 5' and 3' ends of the 1.3 kb transcript in ribonuclease protection studies, and will investigate further the nature of the 3.8 kb transcript. We are also attempting to raise antibodies to the putative Tc1 open reading frame protein using as immunogens both peptides and protein synthesized from an expression vector. We will use these to study expression of Tc1 at the protein level.

Way JC et al. (1985) Worm Breeder's Gazette "progress in cloning the mec-3 gene."

Chalfie M, Way JC

[Worm Breeder's Gazette, 1985]

We used the spontaneous mutant u298 isolated from TR679 to clone at least part of the mec-3 gene by the now-standard 'transposon tagging' method used by Don Moerman to clone unc-22 and Iva Greenwald to clone lin-12. The original mutant was backcrossed to N2 several times, then crossed with unc-24 (0.7 m.u. to the left) and dov-20 (0.3 m.u. to the right). We identified a single Tc1-containing EcoRI fragment which always co-segregated with the mec-3 mutation. From an outcrossed strain, we cloned a Pst-Bam fragment containing the Tc1 insertion into the plasmid pUC8. We also constructed a restriction map of this region of the C. elegans genome. [See Figure 1] A plasmid derivative in which the Tc1 had been excised (pTU1) was used to probe Southern blots of various mutants. All mec-3::Tc1 insertions are at the same site in the same orientation. (As described above, we may only have two independent insertions.) TR679 and a revertant of one of the insertions have the same pattern as N2 for all enzymes tested. The Tc1 elements are identical to the sequenced Tc1 (Rosensweig et al. NAR 11, 4201) in their restriction map as far as we know. (The cloned Tc1 does not have a HindIII site; we don't know about the others.) We can see a faint somatic excision band at the wild-type position in digests of each Tc1 mutant. EMS induced mutations show no change in pattern. We have not yet identified a transcript from this region; nor have John Sulston and Alan Coulson identified a cosmid contig containing our clone (because the cloned DNA is too small). We are doing all the obvious things. Information about the genetic map around mec-3Positions of cosmids. Two cosmids which pick up N2/BO RFLPs were positioned relative to the markers unc-24, mec-3 and dpy-20.[See Figure 2] All 4/4 + mec-3 recombinants derived from eDF18 / + + (BO) heterozygotes had the BO pattern when probed with BO555, so this cosmid maps near or to the left of the right endpoint of eDF18. The CGBP1 cosmid was shown by Don Moerman to contain a hotspot for spontaneous Tc1 insertion. When used as a probe, this cosmid reveals a PstI polymorphism (the polymorphic bands are both >20 kb), which maps as shown, and a cross-hybridizing PstI polymorphic region which maps to the left of unc-24.A third cosmid, BO5I5, sent to us from David Baillie, maps to the right of mec-3. We have not positioned it relative to dpy-20.TR679. When either of the above cosmids is used to examine u298 strains containing this region of chr. IV from TR679, the Bristol pattern is revealed. However, in the course of outcrossing the mec-3::Tc1 insertion described above, it was apparent that there are at least 4 additional Tc1 elements between unc-24 and dpy-20 in this particular TR679 stock. All of these Tc1s are in different sized EcoRI fragments from those in analogously constructed BO/N2 hybrids containing BO DNA around mec-3.