[
Worm Breeder's Gazette,
1990]
Unc-73 affects the longitudinal growth of certain neurons and the excretory cell as well as certain longitudinal cell migrations on the nematode epidermis (Hedgecock et al., Development 100, 365-382, 1987). We previously isolated
unc-73(
ev454) on strain RW7097 and identified a Tc1 on a 5 kb HindIII which cosegregated with this mutation in 3 factor crosses (CSH abstracts, 1989, p. 60). We have now cloned this 5 kb Tc1-containing HindIII fragment, excised the Tc1 with EcoRV, and used the flanking sequence to probe Southern blots of DNAs from
unc-73(
ev454) and from two independently derived spontaneous revertants of
unc-73(
ev454). As expected, this probe hybridized to a 5 kb HindIII fragment on the
unc-73(
ev454) lane and to a 3.4 kb HindIII fragment on the revertant lanes. No polymorphisms were observed in DNA from two EMS-induced alleles of
unc-73 (
e936 and
rh40). We have obtained approximately 250 nucleotides of sequence from both ends of the 5 kb HindIII fragment. One of these ends comprises a GC rich open reading frame which fails to show any significant homology to anything in the NBRF database. We have also identified about a dozen phages from an EMBL-3 N2 genomic library (Nishiwaki and Miwa) which hybridize to our Tc1-flanking probe, but we have not yet characterized any of these. We hope that these will allow Coulson and company to identify the unc- 73-containing contig.
[
Worm Breeder's Gazette,
1984]
We have some preliminary results from our attempts to identify mRNA from
unc-22. Probing Northerns with various lambda clones from the
unc-22 region reveals 2 transcripts. One is about 1.6kb in size and hybridizes only to our left-most clone, which only partially spans the region defined by the insertions. We assume this message is the product of a gene adjacent to
unc-22. A second transcript that hybridizes to the entire set of clones spanning the insertion sites is very large--much larger in fact, than the 6.1kb
unc-54 mRNA. The message is somewhat degraded but we do see a definite band. It is present in RNA prepared from mixed cultures and is at roughly 2-10% of the level of
unc-54 message. Our reasons for believing that this large transcript is the product of
unc-22 are threefold: (1) Tc1 insertions over a 20kb distance can disrupt
unc-22 function, which is at least consistent with the large message seen. (2) We have also compared message levels for this presumed
unc-22 transcript in N2 and
unc-22(
s32), an amber allele, and the
s32 signal is greatly reduced relative to the signal from N2 (see Brown et al., Gene 20:139-144, 1982). (3) Finally, we have used single-stranded M13 clones of opposite orientation from the central region of
unc-22 to probe Northerns of N2 and
s32 RNA. One M13 clone hybridizes strongly to the large transcript from N2 but only weakly to RNA from
s32, while the probe of opposite orientation does not hybridize to anything in either lane. From this last result we predict that
unc-22 is transcribed from right to left on the genetic map. (This is a reversal from what we said at the 1984 GSA meeting). Recent sequencing results show that this direction yields the only continuous open reading frame. A preliminary computer search of DNA and protein sequence libraries using this open reading frame sequence did not reveal any homologous sequences to
unc-22.
[
Worm Breeder's Gazette,
1984]
We are attempting to clone
unc-105 and
rol-6, both of which have been established to have wild-type null phenotypes. By repeated genetic backcrosses, we have constructed strains almost entirely derived from the C. elegans Bristol strain except for a small region of Bergerac chromosome II corresponding to the region to which both
unc-105 and
rol-6 have been mapped. We have localized 29 Bergerac- derived Tc1 elements within this region and have cloned in phage lambda-
gt7 seven that map close to
unc-105 and
rol-6.We have mapped the four clones closest to
unc-105 and
rol-6 with respect to a series of LGII deficiencies. (Deficiency strains and the deficiency map shown below have been kindly provided to us by Chris Sigurdson and Bob Herman. The distance represented on this map corresponds to about two genetic map units, which we estimate to be approximately 600 kb of DNA. ) These clones were localized by Southern hybridization experiments using DNA isolated from Bristol strains heterozygous for different deficiencies and using the phage clones as probes. Strains of genotype mnDf
unc-4/C1
unc-52 were grown on agarose plates for a number of generations, and DNA was isolated from the population of animals present, i.e., from a mixture of deficiency homozygotes (dead eggs and/or dead larvae), wild types and C1 homozygotes. Since C1 homozygotes are sterile and deficiency homozygotes are inviable, we assume that both LGII chromosomes are represented approximately equally in the DNA preparations. We have probed DNA both from N2 and from different deficiency strains with our phage clones. In each case, Southern hybridization experiments have revealed one novel band in addition to those Tc1-containing bands normally seen in Bristol strains; this novel band presumably corresponds to the unique sequence DNA surrounding the Tc1 element in the cloned probe. For cases in which the DNA of a deficiency strain resulted in this band's being of reduced intensity relative to the Tc1- containing bands (and compared to a lane containing N2 DNA), we concluded that the clone used as a probe mapped within that deficiency. The map locations of four of our clones are shown below. [See Figure 1]
[
Worm Breeder's Gazette,
1986]
The accumulation of altered or damaged enzymes in aging organisms, first observed in Turbatrix aceti by Gershon & Gershon, has also been found to occur in mammals. It has been suggested that this accumulation may result from an age-dependent decline in the ability of cells to degrade these abnormal proteins. Sharma et al. [1979] and Prasanna & Lane [1979] reported an age-dependent decline in turnover rates in T. aceti, both for specific enzymes and for global protein. The measurement of protein turnover is fraught with technical difficulties and we know very little about the enzymatic mechanisms involved. One simple hypothesis to explain declining rates of turnover in older animals is that the amount of cellular protease activity also declines with age, leading to a decreased capacity for intracellular proteolysis. We have found that the activities of several lysosomal proteases decline markedly in aging C. vity of the major aspartyl protease cathepsin D declines about 10-fold from its peak at 4 days of age (growth at 25 C; mean lifespan about 12 days) to a minimum at about 10-11 days. Both the total activity per animal and the specific activity (per unit protein) decline. Immunoblot analysis using anti-cathepsin D antibody shows that this activity loss is accompanied by a parallel loss of cathepsin D measured as antigen. Using enzymatic assays after isoelectric focusing of crude extracts, we have also measured the age-dependence of the thiol cathepsins Ce1 and Ce2 (see Sarkis et al ., this issue) which hydrolyze Z-phe-arg- aminomethylcoumarin. From day 3 to day 11, the specific activity of cathepsin Ce2 declines about 8-fold, whereas that of cathepsin Ce1 declines about 2.5-fold. These experiments also revealed a previously undetected enzyme which hydrolyzes the same substrate, but has a different isoelectric point from those of Ce1 and Ce2. On the basis of IEF analysis of purified lysosomes, this new enzyme is nonlysosomal. Its specific activity is essentially independent of the age of the animals. The age-dependent decrease in lysosomal protease activities is quite different from the strong (as much as 100-fold) increase in lysosomal glycosidase and phosphatase activities (Bolanowski et al., 1983). We think it possible that the decrease in protease levels is causally related to the increase in other lysosomal enzyme levels, since the cad-l mutant which is deficient in cathepsin D has abnormally elevated levels of -hexosaminidase and -glucosidase at all ages. It would not be surprising if lysosomal glycosidases were at risk for proteolysis while cohabiting with proteases. The age- dependent declines in lysosomal protease activities are at least consistent with the possibility that there is a simple enzymatic basis for decreasing protein turnover capacity in aging animals. We emphasize, however, that there is still no direct evidence that these Iysosomal enzymes are principally responsible for intracellular protein turnover.
[
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.
[
Worm Breeder's Gazette,
1992]
We reported previously that several X-linked elements that feminize triploid males all contain an octmer sequence which serves, in only one orientation, as a single-stranded DNA-binding site for a protein present in worm nuclear extracts (see Abstracts for 1991 Meeting, pg. 354). We have now purified this binding activity (termed FEBP) and are cloning the gene for this protein. Our purification scheme was simplified by the observation that the sequence-specific ssDNA-binding activity survived boiling. Following removal of denatured proteins by centrifugation, the boiled extract was directly loaded onto an affinity column. The column consisted of a single-stranded oligonucleotide (containing the octamer in correct orientation and derived from the first intron of the
act-4 gene) attached to an agarose matrix. Following elution at high salt the predominant protein visible on Coomassie-stained SDS gels was approx. 30-32 kd, with several other bands both above and below 30 kd also visible. These protein bands were individually cut from preparative SDS gels, eluted, denatured and renatured. Band shift activity was not associated with the predominant 30 kd protein, however, but instead with a single band at approx. 24 kd. This purified and renatured protein demonstrated single-strand specificity, octamer specificity (as assayed by competitions), and, when UV crosslinked to the single-stranded probe, gave the same band on a SDS gel as previously reported (30-32 kd). It was, nonetheless, still of some interest to us to know what the predominant 30 kd protein was, since until the denaturation/renaturation experiments it seemed a likely candidate itself for FEBP. Both proteins were therefore gel-purified following the affinity column and microsequenced (William Lane, Harvard Microchem). 30-32 kd protein: Based on the sequence of one trypsin peptide an unambiguous identification could be made. It is a member of a small family of DNA-binding proteins called Y-box binding proteins and appears to be identical to a Y-box binding protein identified and cloned by Fire and co-workers (WBG 12 (2), pg. 72). This protein was initially identified by virtue of its binding to an upstream regulatory sequence of the
unc-54 gene. Exactly why this protein is binding to our ss-DNA affinity column is unclear, although reports on Y-box binding proteins in several other systems have detected interactions with single-stranded nucleic acids. FEBP: Three different trypsin peptides of 14, 21 and 25 residues respectively have been sequenced. Protein database searches reveal no homologies to known proteins for two of the three peptides; the third peptide shows a small block of homology (7 of 8 or 9 of 14 identities) to the N-terminus region of E. coli SSB, a sequence non-specific single-stranded DNA binding protein. The significance of this result is unclear as this region appears to lie outside of the SSB DNA-binding domain. Genomic PCR as well as RT-PCR cloning using degenerate primers based upon these peptide sequences is underway. In addition, cDNA libraries are being screened with longer degenerate oligonucleotides. Mice have been injected with purified FEBP (as well as, separately, with the 30 kd protein) and preliminary ELISA results with purified antigens indicate immunoreactivity in the tested sera; fusions should follow the next boost or two. Rabbit antisera await either injection with coupled peptides or with cDNA expression products.
[
Worm Breeder's Gazette,
1988]
Borrowing yet again from yeast molecular biology, the following procedure has been used to isolate clean, intact RNA from small quantities of worms. This saves the time and effort of growing up, and grinding in liquid nitrogen, large liquid cultures. This procedure should make it possible to process easily 10 to 20 stocks in parallel for screening by Northerns. Yields are ~200 g of clean RNA from 4x9 cm 'high yield' plates (see below), or about 2 g of clean RNA from a single, 5 cm streaked plate. Mini-prep. Wash worms off 4 plates (5 cm) that are just clearing. If you want, the worms can be incubated in S-media for 30 min to digest whatever they have in their gut, and then purified by standard sucrose flotation in a 5ml tube. Resuspend the worm pellet in 0.5 ml of liquid. (Use any RNAase-free, solvent resistant, sterile tube that is big enough. I use Falcon 2098 50ml disposable polypropylene tubes.) Add 2ml GuEST buffer and 2ml PCI (see below), and then 6g glass beads. Vortex on high speed, using a regular bench vortex, for 2 min at room temp. Draw the liquid off the beads with a pipetman into 4x1.5 ml eppendorf tubes, rinse the beads with 0.5ml of PCI, and add this to the microfuge tubes. Microfuge for 2 min. Transfer the aqueous layer to fresh tubes, add 1/10 vol 3M NaAcetate, pH 6.0, and re-extract with PCI. Spin, transfer the aqueous phase (leaving the interface, if any, behind), and precipitate the RNA by the addition of 2 vol of 100% ethanol. -20 C for 20 min, spin in microfuge 10 min, decant, and wash pellet with 80% ethanol. Dry, and dissolve the pellet in 0.3ml of dddH20 (see below). Add 0.9ml of 4M NaAcetate (diethylpyrocarbonate ( depc) treated), and let sit at least 5 hr at 4 C. Spin in microfuge 10 min, discard supernatant. Dissolve the pellet in 0.1ml dddH2O, add 5 l 3M NaAcetate, and 210 l 100% ethanol. Precipitate at -20 C for 20 min, spin, wash with 80%, and dissolve the final pellet in 50 l dddH2O. Dilute 2 l to 1ml with dddH20, read A260 and multiply the absorbance reading by 20 to get the RNA concentration in mg/ml. p(A)+ selection, using poly(U) Sepharose (Jacobson [1987] Meth. Enz. 152:254261), should give 2-4 g of p(A)+ RNA, although using total RNA has given me good signals on a Northern with an actin probe. If you are in a hurry, you can skip the NaAcetate precipitation step, since large MW DNA won't transfer upon Northern Blotting. Micro-prep. Wash the worms off a single 5 cm plate that is just clearing. Repeat the above procedure, using 50 l worms, 100 l GuEST buffer, 100 l PCI and 0.5g glass beads. Rinse beads with 50 l PCI. Do the salt precipitation of RNA in 30 l by adding 90 l of 4M Na Acetate, and dissolve the final pellet in 10 l. Frozen Worms. I have used this procedure, successfully, to prepare poly(A)+ RNA from frozen worms. Washed worms were suspended in 0.1M NaCl, quick-frozen in liquid nitrogen, and kept at -70 C for 1 month. Use 4ml of GuEST, 4ml of PCI and 12g of beads per ml of worms. Thaw the tube just until the frozen plug of worms can be removed, and drop the plug into the GuEST/PCI. Proceed as with the mini-prep, although you may want to vortex a bit longer. Buffers. GuEST (From M. Goedert) Add 245ml sterile distilled water to 200g Guanidine isothiocyanate (BRL Ultra Pure). Add 21ml 1M Tris pH 7.4, and 42ml 100mM EDTA. Heat gently to dissolve. Add 9ml Sarkosyl, and 4.2ml -mercaptoethanol. Bring volume to 420ml with sterile distilled water. Filter through a sterile Nalgene filter, and store at 4 C. PCI. Phenol:Chloroform:Isoamyl alcohol, 25:24:1 Glass Beads. 0.3 to 0.4 mm diam (although I haven't tried other sizes). I borrowed mine from J. Kilmartin, but apparently BDH and Sigma sell them. They can be acid washed, baked and re-used. dddH20. Add depc (0.07% v/v) to sterile, double-distilled water, shake for 10 min, and then autoclave. 'High Yield' plates. (From A. Spence) Add a drop of 20% glucose to a large NGM plate, and then spread the plate with a wild-type bacteria such as NA22. Let sit overnight before adding worms. HINTS: Wear gloves, use only sterile tubes and pipette tips, and generally treat the RNA as you would HIV, and you should have no problems. A quick method for checking the quality of your RNA is on a 0.8% agarose minigel. Denature the RNA for 10 min at 65 C, chill on ice and add sterile glycerol/dye/buffer. Run 2 g RNA/lane in a RNAase free gel box with standard gel buffer, ~7 V/cm for 40 min. EtBr stain 5', photograph. You should see two tight, rRNA bands, with no high MW DNA visible. Avoid using old plates, where the worms have started burrowing, as the softened agar tends to wash off with the worms, and contaminate the RNA. In these cases a small-scale sucrose flotation might be useful to clean up the worms, but I haven't yet tried this.